摘要
目的：通过生物信息技术对数据库芯片结果进行筛选,以发现可能与慢性疼痛(神经病理性疼痛)发病相关的新致病基因,并对所过滤的差异基因表达谱进行分析。同时构建大鼠脊神经结扎(SNL)模型,观察大鼠疼痛行为学变化,并检测所获致病基因在损伤及非损伤背根神经节(DRG)的表达情况及在不同神经元中的分布,以验证生物信息学筛选基因的可行性,并探讨所获致病基因对神经病理性疼痛发病的可能影响机制。
方法：1、从GEO数据库中搜索并下载与神经病理性疼痛相关的芯片数据,应用BRB ArrayTools软件对数据进行标准化、过滤及差异基因比较,通过基因功能挖掘平台挖掘文献,并与已有Meta分析比对筛选出未在SNL模型损伤及非损伤侧DRG中发现的致病基因,同时,应用DAVID6.7数据库对非损伤DRG中的差异基因进行分析,探讨非损伤DRG中可能发生的病理生理变化。
2、将65只大鼠随机分为正常组(n=15)、Sham组(n=25)及SNL组(n=25),SNL组建立大鼠左侧SNL模型,Sham组仅暴露左侧L5脊神经而不结扎,正常组未作处理。术前1天及术后1、4、7、10、13、16、19、22、25、28天测定各组大鼠左后足机械痛阈变化情况,术后3、7、14、21、28天分批处死,取术侧及健侧L4、L5DRG,采用RT-PCR及Western-Blot技术分别对生物信息学方法所获致病基因的mRNA及蛋白表达变化水平进行检测。
3、将36只大鼠随机分为正常组(n=9)、Sham组(n=9)及SNL组(n=18),SNL组建立大鼠左侧SNL模型,Sham组仅暴露左侧L5脊神经而不结扎,正常组未作处理。术前1天及术后1、3、5、7、9、11、14天测定各组大鼠左后足机械痛阈变化情况,术后3、7、14天分批灌注处死,取术侧L4、L5DRG,采用免疫荧光技术检测生物信息学方法所获致病基因在NF200、IB4和CGRP免疫阳性DRG神经元中的表达变化情况。
结果：1、采用生物信息技术共获得123条同时在损伤及非损伤DRG中差异表达的基因,经过进一步文献挖掘发现34条与神经病理性疼痛发病相关,其中20条基因未见报道NSF在已有Meta分析中被认定为与疼痛发生具有最明显联系,此外,在非损伤DRG中基因差异表达主要导致了离子通道及离子运输、神经元损伤、免疫应答、囊泡转运等方面的变化。
2、术前三组大鼠机械痛阈无显著差异(P>0.05),术后SNL组与空白组及Sham组相比,机械痛阈显著下降(P<0.05),同时术侧后肢抬起、足趾并拢、足底轻度外翻,而健侧及其它两组大鼠无明显变化。
3、术后各时间点RT-PCR及Western-Blot结果均显示SNL组大鼠术侧L4、L5DRG内NSF的mRNA及蛋白水平较健侧、正常组及Sham组L4、L5DRG显著下降(P<0.05)。
4、免疫荧光结果显示NSF在正常大鼠三种亚型DGR中分布广泛,NSF(?)日性神经元主要分布于中小神经元。NSF在SNL大鼠IB4及CGRP免疫阳性神经元中表达下降,同时NSF的表达逐渐由中小神经元转向大中神经元。
结论：1、利用生物信息技术能够寻找到与神经病理性疼痛发病相关的致病基因,并能通过分子生物学和组织学试验得到验证,提示其可以作为寻找疾病致病基因的一种有效方法。
2、大鼠脊神经结扎后NSF的表达下调可能与神经病理性疼痛的发生发展有关。
3、生理状态下NSF可能参与DRG各亚型神经元中无害及有害感觉信息的传递,而脊神经结扎后IB4及CGRP阳性神经元中的NSF表达变化可能在神经病理性疼痛的发生发展中起着更为重要的作用。
Objective:Using bioinformatics technology to mine the new pathogenic genes and analyze the expression profile of differential expressed genes. Besides, to observe the time-dependent variation tendency of pain behavior and investigate the mRNA and protein expression level as well as the distribution in different DRG neuron subtypes, and to verify the possibility of the gene mining method based on bioinformatics technology and the potential effect of mined new pathogenic gene in the neuropathic pain.
Methods:1. The microarray data was downloaded from online database GEO. The data were normalized, filtered and compared by BRB ArrayTools. In order to make sure that the expression of obtained new pathogenic genes was not reported in the L4and L5DRG of SNL model, the differential genes were detected in data mining platforms and compared with the existing Meta analysis research. Meanwhile, DAVID6.7database was applied for the analysis of differential expressed genes and related physiopathologic change in uninjured DRG.
2.65adult male SD rats were randomly divided into3groups: Normal group (n=15), Sham group (n=25) and SNL group (n=25). For the rats of SNL group, left L5spinal nerve was ligated, while L5spinal nerve was only exposed without ligation for the rats of sham group, and the normal group was not treated by any surgery. The mechanical threshold values were measured before the surgery and1,4,7,10,13,16,19,22,25and28days after SNL surgery. The rats were scarified respectively at3,7,14,21and28days after SNL surgery, and the L4and L5DRG at both sides were obtained to verify the expression level of new pathogenic genes by RT-PCR and Western Blot experiments.
3.36adult male SD rats were randomly divided into3groups: Normal group (n=9), Sham group (n=9) and SNL group (n=18). For the rats of SNL group, the left L5spinal nerve was ligated, while L5spinal nerve was only exposed without ligation for the rats of sham group and the normal group was not treated by any surgeries. The mechanical threshold values were measured1day before the surgery and1,3,5,7,9,11and14days after SNL surgery. The rats were then perfused and scarified respectively at3,7and14days after SNL surgery, and the L4and L5DRG at the operative side were obtained to verify the distribution of new pathogenic gene in different DRG neuron subtypes by immunofluorescence technology.
Results:1.123genes which expressed differentially in L4and L5DRG were obtained by bioinformatics technology. After further literature mining,34genes were related with the neuropathic pain, and expressions of20genes in them were not reported. NSF has the strong and correlational evidence with neuropathic pain based on the exsited Meta analysis. Besides, the differentially expressed genes induced the change of ion channel, ion transportation, neuron damage, immune response and vesicle transportation in the uninjured L4DRG.
2. The50%mechanical paw withdrawal threshold (50%MWT) values were not significantly different among3groups before SNL surgery (P>0.05). After the SNL surgery,50%MWT values of SNL group rats decreased significantly compared with Normal and Sham group (P<0.05). Meanwhile, some behavior changes such as everted foot and closed toes were observed on the rats of SNL group.
3. Through the analysis of RT-PCR and Western Blot, the mRNA and protein expression level of NSF decreased significantly in ipsilateral L4and L5DRG of SNL rats while compared with the DRGs in contralateral sides, Normal and Sham group.
4. In the normal rats, the NSF was distributed widely in NF200-, IB4-and CGRP-positive DRG neurons. And most of the NSF positive neurons were small and medium size neurons. In the SNL rats, the NSF positive rates in IB4and CGRP positive neurons decreased significantly (P<0.05), and the area distribution of NSF positive neurons moved to the large and medium size.
Conclusions:1. The bioimformatics technology can effectively mine the potential pathogenic genes of neuropathic pain, which can be verified by further molecular biology and histology experiments.
2. The significant decreases of NSF mRNA and protein levels in L4and L5DRG of SNL rats as well as the persistent pain-related behaviors were observed, which can indicate that the expression change of NSF plays a role in the neuropathic pain.
3. The wide distribution of NSF in normal DRG neurons implies that NSF can regulate the transmission of both innocuous and noxious sensory information. After SNL surgery, the area of NSF positive neurons were changed, and the positive rates in IB4and CGRP positive neurons were deceased, which implies a more important role of the NSF expression in IB4and CGRP positive neurons.
引文
[1]Bridges D, Thompson SW, Rice AS. Mechanisms of neuropathic pain [J]. Br J Anaesth.2001; 87(1):12-26.
[2]Vallejo R, Tilley DM, Vogel L, et al. The role of glia and the immune system in the development and maintenance of neuropathic pain [J]. Pain Pract.2010; 10(3):167-184
[3]Woolf CJ, Salter MW. Neuronal plasticity:increasing the gain in pain [J]. Science,2000; 288(5472):1765-1769.
[4]Yaksh TL, Hua XY, Kalcheva I, et al. The spinal biology in humans and animals of pain states generated by persistent small afferent input [J].Proc Natl Acad Sci U S A.1999; 96(14):7680-7686.
[5]LaCroix-Fralish ML, Austin JS, Zheng FY, et al. Patterns of pain: Meta-analysis of microarray studies of pain [J]. Pain.2011; 152(8):1888-1898.
[6]Yang ZM, Chen WW, Wang YF. Gene expression profiling in gastric mucosa from Helicobacter pylori-infected and uninfected patients undergoing chronic superficial gastritis [J]. PLoS One.2012; 7(3):e33030.
[7]Li Y, Dorsi MJ, Meyer RA, et al. Mechanical hyperalgesia after an L5 spinal nerve lesion in the rat is not dependent on input from injured nerve fibers [J].Pain. 2000; 85(3):493-502.
[8]Dolly JO, O'Connell MA. Neurotherapeutics to inhibit exocytosis from sensory neurons for the control of chronic pain [J]. Curr Opin Pharmacol.2012; 12(1):100-108
[9]Sun T, Xiao HS, Zhou PB, et al. Differential expression of synaptoporin and synaptophysin in primary sensory neurons and up-regulation of synaptoporin after peripheral nerve injury [J]. Neuroscience.2006; 141(3):1233-1245.
[10]Schmidtko A, Del Turco D, Coste O, et al. Essential role of the synaptic vesicle protein synapsin II in formalin-induced hyperalgesia and glutamate release in the spinal cord [J]. Pain.2005; 115(1-2):171-181.
[11]Camprubi-Robles M, Planells-Cases R, Ferrer-Montiel A. Differential contribution of SNARE-dependent exocytosis to inflammatory potentiation of TRPV1 in nociceptors [J]. FASEB J.2009; 23(11):3722-3733.
[12]Zhao C, Matveeva EA, Ren Q, et al. Dissecting the N-ethylmaleimide-sensitive factor:required elements of the N and D1 domains [J]. J Biol Chem.2010; 285(1):761-772.
[13]Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence [J]. Physiol Rev.2001; 81(1):299-343.
[14]Li H, Zhou Y, Kang L, et al. Single and repeated morphine administrations differently regulate expression of N-ethylmaleimide-sensitive factor gene in the rat brain [J]. Neuroreport.2006 23; 17(1):71-74.
[15]Yin S, Guan Z, Tang Y, et al. Abnormal expression of epilepsy-related gene ERG1/NSF in the spontaneous recurrent seizure rats with spatial learning memory deficits induced by kainic acid [J]. Brain Res.2005; 1053(1-2):195-202.
[16]Katano T, Furue H, Okuda-Ashitaka E, et al. N-ethylmaleimide-sensitive fusion protein (NSF) is involved in central sensitization in the spinal cord through GluR2 subunit composition switch after inflammation [J]. Eur J Neurosci.2008; 27(12):3161-3170.
[17]Hammond DL, Ackerman L, Holdsworth R, et al. Effects of spinal nerve ligation on immunohistochemically identified neurons in the L4 and L5 dorsal root ganglia of the rat [J]. J Comp Neurol.2004; 475(4):575-589.
[18]Hunt SP, Mantyh PW. The molecular dynamics of pain control [J]. Nat Rev Neurosci.2001; 2(2):83-91
[19]Meyer RA, Ringkamp M. A role for uninjured afferents in neuropathic pain [J]. Acta Physiologica Sinica.2008; 60(5):605-609.
[20]Takasu K, Sakai A, Hanawa H, et al. Overexpression of GDNF in the uninjured DRG exerts analgesic effects on neuropathic pain following segmental spinal nerve ligation in mice [J]. J Pain.2011; 12(11):1130-1139.
[21]von Schack D, Agostino MJ, Murray BS, et al. Dynamic changes in the microRNA expression profile reveal multiple regulatory mechanisms in the spinal nerve ligation model of neuropathic pain [J]. PLoS One.2011; 6(3):e17670.
[22]Simon R, Lam A, Li MC, et al. Analysis of gene expression data using BRB-ArrayTools. [J]. Cancer Inform.2007,4(3):11-17.
[23]Vega-Avelaira D, Geranton SM, Fitzgerald M. Differential regulation of immune responses and macrophage/neuron interactions in the dorsal root ganglion in young and adult rats following nerve injury [J]. Mol Pain.2009; 5:70.
[24]Ashburner M, Ball CA, Blake JA, et al. Gene ontology:tool for the unification of biology. The Gene Ontology Consortium [J]. Nat Genet.2000; 25(1):25-29.
[25]Kanehisa M, Goto S. KEGG:kyoto encyclopedia of genes and genomes [J]. Nucleic Acids Res.2000; 28(1):27-30.
[26]Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources [J]. Nature Protoc.2009; 4(1):44-57.
[27]Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists [J]. Nucleic Acids Res.2009; 37(1):1-13.
[28]王桂平,叶云,郑文岭,等.Toppgene筛选肺腺癌候选疾病基因[J].中国肺癌杂志,2010,13(4)：282-286.
[29]Stelzer G, Dalah I, Stein TI, et al. In-silico human genomics with GeneCards [J]. Hum Genomics.2011; 5(6):709-717.
[30]Chen J, Aronow BJ, Jegga AG Disease candidate gene identification and prioritization using protein interaction networks [J]. BMC Bioinformatics.2009; 10:73.
[31]Liekens AM, De Knijf J, Daelemans W, et al. BioGraph:unsupervised biomedical knowledge discovery via automated hypothesis generation [J]. Genome Biol.2011;12(6):R57.
[32]Davis S, Meltzer PS. GEO query:a bridge between the Gene Expression Omnibus (GEO) and BioConductor [J]. Bioinformatics.2007; 23(14):1846-1847.
[33]Barrett T, Edgar R. Gene expression omnibus:microarray data storage, submission, retrieval, and analysis [J]. Methods Enzymol.2006; 411:352-369.
[34]李铁求,冯春琼,邹亚光,等.基于文献挖掘的雄激素非依赖型前列腺癌特异表达基因的生物信息学分析[J].中华男科学杂志.2009,15(12)：1102-1107.
[35]李飞,梁中锟,荆玉明,等.基于BRB—ArrayTools和GATHER工具对膀胱癌相关基因的挖掘及生物信息学分析[J].临床泌尿外科杂志.2011,26(11)：852-855.
[36]Iwamoto T, Bianchini G, Booser D, et al. Gene pathways associated with prognosis and chemotherapy sensitivity in molecular subtypes of breast cancer [J]. J Natl Cancer Inst.2011,103(3):264-272..
[37]Chen YA, Scheller RH. SNARE-mediated membrane fusion [J]. Nat Rev Mol Cell Biol.2001; 2(2):98-106.
[38]Miyagawa S,Kubo T, Matsunami K, et al. Delta-short consensus repeat 4 decay accelerating factor (DAF:CD55) inhibits complemen-t mediated cytolysis but not NK cell mediated cytolysis [J]. J Immunol,2004,173(6):3945-3952.
[39]Tosello AC, Mary F, Amiot M, et al. Activation of T cells via CD55: recruitment of early components of the CD3 TCR pathway is required for IL-2 secretion [J]. J Inflamm,1998,48(1):13-27.
[40]Kolev M, Towner L, Donev R. Complement in cancer and cancer immunotherapy [J]. Arch Immunol Ther Exp (Warsz).2011; 59(6):407-419.
[41]Vague P, Coste TC, Jannot MF, et al. C-peptide, Na+,K(+)-ATPase, and diabetes [J]. Exp Diabesity Res.2004;5(1):37-50.
[42]张雪冰,白然,杜建玲,等.Na/K ATP酶活性及其ATP1A1基因多态性与2型糖尿病周围神经病变的相关性[J].中国糖尿病杂志.2007；15(4)：222-224.
[43]Fukuoka T, Yamanaka H, Kobayashi K, et al. Re-evaluation of the phenotypic changes in L4 dorsal root ganglion neurons after L5 spinal nerve ligation [J]. Pain.2012; 153(1):68-79.
[44]Zhang XF, Zhu CZ, Thimmapaya R, et al. Differential action potentials and firing patterns in injured and uninjured small dorsal root ganglion neurons after nerve injury [J]. Brain Res.2004; 1009(1-2):147-158.
[45]Chahine M, Ziane R, Vijayaragavan K, et al. Regulation of Na v channels in sensory neurons [J]. Trends Pharmacol Sci.2005; 26(10):496-502.
[46]Berta T, Poirot O, Pertin M, et al. Transcriptional and functional profiles of voltage-gated Na(+) channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain [J]. Mol Cell Neurosci.2008; 37(2):196-208.
[47]Shortland PJ, Baytug B, Krzyzanowska A, et al. ATF3 expression in L4 dorsal root ganglion neurons after L5 spinal nerve transaction [J]. Eur J Neurosci. 2006;23(2):365-373.
[48]Djouhri L, Koutsikou S, Fang X, et al. Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors [J]. J Neurosci.2006; 26(4):1281-1292.
[49]Clark AK, Yip PK, Grist J, et al.. Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain. Proc. Natl. Acad. Sci. U. S. A.2007; 104(25): 10655-10660.
[50]Schomberg D, Olson JK. Immune responses of microglia in the spinal cord: contribution to pain states [J]. Exp Neurol.2012; 234(2):262-270.
[51]Ji RR, Xu ZZ, Wang X, et al. Matrix metalloprotease regulation of neuropathic pain [J]. Trends Pharmacol Sci.2009; 30(7):336-340.
[52]Levin ME, Jin JG, Ji RR, et al. Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain [J]. Pain. 2008; 137(1):182-201.
[53]Li M, Peake PW, Charlesworth JA, et al. Complement activation contributes to leukocyte recruitment and neuropathic pain following peripheral nerve injury in rats [J]. Eur J Neurosci.2007; 26(12):3486-3500.
[54]王金保,聂发传,王姝媛,等.脊髓背角补体异常活化与神经病理性疼痛关系的实验研究[J].国际麻醉学与复苏杂志.2009,30(3)：217-222.
[55]Andratsch M, Mair N, Constantin CE, et al. A key role for gp130 expressed on peripheral sensory nerves in pathological pain [J]. J Neurosci.2009; 29(43):13473-13483.
[56]Geppert M, Sudhof TC. RAB3 and synaptotagmin:the yin and yang of synaptic membrane fusion [J]. Annu Rev Neurosci.1998; 21(2);75-95.
[57]Garry EM, Moss A, Rosie R, et al. Specific involvement in neuropathic pain of AMPA receptors and adapter proteins for the GluR2 subunit [J]. Mol Cell Neurosci. 2003;24(1):10-22.
[58]Kawasaki F, Ordway RW. Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants [J]. Neuroscience,2009,106(34):14658-14663.
[59]Leenders AG, Sheng ZH. Modulation of neurotransmitter release by the second messenger-activated protein kinases:implications for presynaptic plasticity [J]. Pharmcol Ther,2005,105(1):69-84.
[60]Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat [J]. Pain.1992; 50(3):355-363.
[61]Kim KJ, Yoon YW, Chung JM. Comparison of three rodent neuropathic pain models [J]. Exp Brain Res.1997; 113(2):200-206.
[62]刘国凯,黄宇光,罗爱伦.神经病理性疼痛动物模型及其评价[J].中国临床药理学与治疗学,2005,10(6)：601-603.
[63]Hogan Q, Sapunar D, Modric-Jednacak K, et al. Detection of neuropathic pain in a rat model of peripheral nerve injury [J]. Anesthesiology.2004; 101(2):476-487.
[64]Engle MP, Gassman M, Sykes KT, et al. Spinal nerve ligation does not alter the expression or function of GABAB receptors in spinal cord and dorsal root ganglia of the rat [J]. Neuroscience,2006,138(4):1277-1287.
[65]Engle MP, Merrill MA, Marquez De Prado B, et al. Spinal nerve ligation decreases γ-aminobutyric acidB receptors on specific populations of immunohistochemically identified neurons in L5 dorsal root ganglion of the rat [J]. J Comp Neurol.2012; 520(8):1663-1677.
[66]姜雨鸽,徐龙河,张宏.背根神经节在疼痛机制和治疗中的作用[J].国际药学研究杂志.2008,35(1)：18-22.
[67]Zhang C, Zhou Z. Ca(2+)-independent but voltage-dependent secretion in mammalian dorsal root ganglion neurons [J]. Nat Neurosci.2002; 5(5):425-430.
[68]Liu T, Shang SJ, Liu B, et al. Two distinct vesicle pools for depolarization-induced exocytosis in somata of dorsal root ganglion neurons [J]. J Physiol.2011; 589(Pt 14):3507-3515
[69]Zhang X, Aman K, Hokfelt T. Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy [J]. J Comp Neurol.1995; 352(4):481-500.
[70]Jahn R, Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles [J]. Nature.2012; 490(7419):201-207.
[71]Dixon WJ. Efficient analysis of experimental observations [J]. Annu Rev Pharmacol Toxicol.1980;20:441-462.
[72]Siidhof TC, Rizo J. Synaptic Vesicle Exocytosis [J]. Cold Spring Harb Perspect Biol 2011; 3(12):a005637.
[73]Noel J, Ralph GS, Pickard L, et al. Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF-dependent mechanism [J]. Neuron.1999, 23(2),365-376.
[74]Lledo PM, Zhang X, Sudhof TC, et al. Postsynaptic membrane fusion and long-term potentiation [J]. Science.1998,279(5349),399-403.
[75]Ohnami S, Kato A, Ogawa K, et al. Effects of milnacipran, a 5-HT and noradrenaline reuptake inhibitor, on C-fibre-evoked field potentials in spinal long-term potentiation and neuropathic pain [J]. Br J Pharmacol.2012; 167(3):537-547.
[76]Lee DZ, Chung JM, Chung K, et al. Reactive oxygen species (ROS) modulate AMPA receptor phosphorylation and cell-surface localization in concert with pain-related behavior [J]. Pain.2012; 153(9):1905-1915.
[77]Sanyal S, Krishnan KS. Genetic modifiers of comatose mutations in Drosophila:insights into neuronal NSF (N-ethylmaleimide-sensitive fusion factor) functions [J]. J Neurogenet 2012; 26(3-4):348-59.
[78]Zeilhofer HU. Prostanoids in nociception and pain [J]. Biochem Pharmacol. 2007; 73(2):165-174.
[79]Fukuoka T, Tokunaga A, Kondo E, et al. Change in mRNAs for neuropeptides and the GABA(A) receptor in dorsal root ganglion neurons in a rat experimental neuropathic pain model [J]. Pain.1998; 78(1):13-26.
[80]Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw [J]. J Neurosci Methods.1994; 53(1):55-63.
[81]Ramakrishnan NA, Drescher MJ, Drescher DG The SNARE complex in neuronal and sensory cells [J]. Mol Cell Neurosci.2012; 50(1):58-69.
[82]Study RE, Krai MG. Spontaneous action potential activity in isolated dorsal root ganglion neurons from rats with a painful neuropathy [J]. Pain.1996; 65(2-3):235-242.
[83]Robertson B, Perry MJ, Lawson SN. Populations of rat spinal primary afferent neurons with choleragenoid binding compared with those labelled by markers for neurofilament and carbohydrate groups:a quantitative immunocytochemical study [J]. J Neurocytol.1991,20(5):387-395.
[84]Morris JL, K6nig P, Shimizu T, et al., Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate [J]. J Comp Neurol.2005; 483(1):1-16.
[85]Yu W, Kawasaki F, Ordway RW. Activity-dependent interactions of NSF and SNAP at living synapses [J]. Mol Cell Neurosci.2011; 47(1):19-27
[86]Morikawa Y, Nishida H, Misawa K, et al. Induction of synaptosomal-associated protein-23 kD (SNAP-23) by various cytokines [J]. Blood. 1998; 92(1):129-135.
[87]Sφrensen JB, Nagy Q Varoqueaux F, et al. Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23 [J]. Cell.2003; 114(1):75-86.
[88]Luo H, Cheng J, Han JS, et al. Change of vanilloid receptor 1 expression in dorsal root ganglion and spinal dorsal horn during infammatory nociception induced by complete Freund's adjuvant in rats [J]. Neuroreport.2004; 15(4):655-658.
[89]Gangadharan V, Agarwal N, Brugger S, et al. Conditional gene deletion reveals functional redundancy of GABAB receptors in pe-ripheral nociceptors in vivo [J]. Mol Pain.2009; 5:68.
[1]Yoon TY, Shin YK. Progress in understanding the neuronal SNARE function and its regulation [J]. Cell Mol Life Sei.2009; 66(3):460-469.
[2]Ramakrishnan NA, Drescher MJ, Drescher DG. The SNARE complex in neuronal and sensory cells [J]. Mol Cell Neurosci.2012; 50(1):58-69.
[3]Chen YA, Scheller RH. SNARE-mediated membrane fusion [J]. Nat Rev Mol Cell Biol.2001;2(2):98-106.
[4]Sollner T, Bennett MK, Whiteheart SW, et al. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion [J]. Cell.1993; 75(3):409-418.
[5]Jahn R, Lang T, Sudhof TC. Membrane fusion [J]. Cell. 2003;112(4):519-533.
[6]Duman JG, Forte JG What is the role of SNARE proteins in membrane fusion? [J]. Am J Physiol Cell Physiol.2003; 285(2):C237-249.
[7]Jahn R, Siidhof TC. Membrane fusion and exocytosis [J]. Annu Rev Biochem. 1999; 68:863-911.
[8]Hu SH, ChristieMP, Saez NJ, et al. Possible roles forMunc18-1 domain 3a and Syntaxinl N-peptide and C-terminal anchor in SNARE complex formation [J]. Proc Natl Acad Sci U S A.2011; 108(3):1040-1045.
[9]Yang B, Gonzalez L Jr, Prekeris R, et al. SNARE interactions are not selective. Implications for membrane fusion specificity [J]. J Biol Chem.1999; 274(9):5649-5653.
[10]Hayashi T, McMahon H, Yamasaki S, et al. Synaptic vesicle membrane fusion complex:action of clostridial neurotoxins on assembly [J]. EMBO J.1994; 13(21):5051-5061.
[11]Laage R, Rohde J, Brosig B, et al. A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins [J]. J Biol Chem.2000; 275(23):17481-17487.
[12]Fasshauer D, Sutton RB, Brunger AT, et al. Conserved structural features of the synaptic fusion complex:SNARE proteins reclassified as Q-and R-SNAREs [J]. Proc Natl Acad Sci U S A.1998; 95(26):15781-15786.
[13]van den Bogaart G, Holt MG, Bunt G, et al. One SNARE complex is sufficient for membrane fusion [J]. Nat Struct Mol Biol.2010; 17(3):358-364.
[14]Burre J, Sharma M, Tsetsenis T, et al. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro [J]. Science.2010; 329(5999):1663-1667.
[15]Siidhof TC, Rizo J. Synaptic vesicle exocytosis [J]. Cold Spring Harb Perspect Biol.2011; 3(12):a005637.
[16]Littleton JT, Chapman ER, Kreber R, et al. Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly [J]. Neuron.1998; 21(2):401-413.
[17]Chen YA, Scales SJ, Patel SM, et al. SNARE complex formation is triggered by Ca2+and drives membrane fusion [J]. Cell.1999; 97(2):165-174.
[18]Shi L, Shen QT, Kiel A, et al. SNARE proteins:one to fuse and three to keep the nascent fusion pore open [J]. Science.2012; 335(6074):1355-1359.
[19]Sinha R, Ahmed S, Jahn R, et al. Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses [J]. Proc Natl Acad Sci U S A.2011; 108(34):14318-14323.
[20]Weber JP, Reim K, Sφrensen JB. Opposing functions of two sub-domains of the SNARE-complex in neurotransmission [J]. EMBO J.2010; 29(15):2477-2490.
[21]Wong JL, Koppel DE, Cowan AE, et al. Membrane hemifusion is a stable intermediate of exocytosis [J]. Dev Cell.2007; 12(4):653-659.
[22]Fang Q, Berberian K, Gong LW, et al. The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics [J]. Proc Natl Acad Sci U S A.2008; 105(40):15388-15392.
[23]Groffen AJ, Martens S, Diez Arazola R, et al. Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release [J]. Science.2010; 327(5973):1614-1618.
[24]Carr CM, Rizo J. At the junction of SNARE and SM protein function [J]. Curr Opin Cell Biol.2010,22(4):488-495.
[25]Han X, Jackson MB. Structural transitions in the synaptic SNARE complex during Ca2+-triggered exocytosis [J]. J Cell Biol.2006; 172(2):281-293.
[26]Tang J, Maximov A, Shin OH, et al. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis [J]. Cell,2006; 126 (6):1175-1187
[27]Yang X, Kaeser-Woo YJ, Pang ZP, et al. Complexin clamps asynchronous release by blocking a secondary Ca2+sensor via its accessory a helix [J]. Neuron. 2010; 68(5):907-920.
[28]May AP, Whiteheart SW, Weis WI. Unraveling the mechanism of the vesicle transport ATPase NSF, the N-ethylmaleimide-sensitive factor [J]. J Biol Chem.2001; 276(25):21991-21994.
[29]Chen X, Tomchick DR, Kovrigin E, et al. Three-dimensional structure of the complexin/SNARE complex. Neuron.; 33(3):397-409.
[33]Dulubova Ⅰ, Khvotchev M, Liu S, et al. Muncl8-1 binds directly to the neuronal SNARE complex [J]. Proc Natl Acad Sci U S A.2007; 104(8):2697-2702.
[28]Rizzoli SO, Betz WJ. The structural organization of the readily releasable pool of synaptic vesicles [J]. Science.2004; 303(5666):2037-2039.
[29]Li F, Pincet F, Perez E, et al. Energetics and dynamics of SNAREpin folding across lipid bilayers [J]. Nat Struct Mol Biol.2007; 14(10):890-896.
[30]Arthur CP, Dean C, Pagratis M, et al. Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons [J]. Neuroscience.2010; 167(1):135-142.
[31]Condliffe SB, Corradini I, Pozzi D, et al. Endogenous SNAP-25 regulates native voltage-gated calcium channels in glutamatergic neurons [J]. J Biol Chem. 2010; 285(32):24968-24976.
[32]Goodyear RJ, Legan PK, Christiansen JR, et al. Identification of the hair cell soma-1 antigen, HCS-1, as otoferlin [J]. J Assoc Res Otolaryngol.2010; 11(4):573-586.
[33]Giraudo CG, Eng WS, Melia TJ, et al. A clamping mechanism involved in SNARE-dependent exocytosis [J]. Science.2006; 313(5787):676-680.
[34]Tobaben S, Thakur P, Fernandez-Chacon R, et al. A trimeric protein complex functions as a synaptic chaperone machine [J]. Neuron.2001; 31(6):987-999.
[35]Sharma M, Burre J, Sudhof TC. CSPa promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity [J]. Nat Cell Biol.2011; 13(1):30-39.
[36]Fernandez-Chacon R, Wolf el M, Nishimune H, et al. The synaptic vesicle protein CSP alpha prevents presynaptic degeneration [J]. Neuron.2004; 42(2):237-251.
[37]Galvin JE, Lee VM, Trojanowski JQ. Synucleinopathies:Clinical and pathological implications [J]. Arch Neurol.2001,58(2):186-190
[38]Chandra S, Gallardo G, Fernandez-Chacon R, et al. Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration [J]. Cell.2005; 123(3):383-396.
[39]Eriksen JL, Przedborski S, Petrucelli L. Gene dosage and pathogenesis of Parkinson's disease [J]. Trends Mol Med.2005; 11(3):91-96.
[40]Young CE, Arima K, Xie J, et al. SNAP-25 deficit and hippocampal connectivity in schizophrenia [J]. Cereb Cortex.1998; 8(3):261-268.
[41]Guerini FR, Bolognesi E, Chiappedi M, et al. SNAP-25 single nucleotide polymorphisms are associated with hyperactivity in autism spectrum disorders [J]. Pharmacol Res.2011; 64(3):283-288.
[42]Etain B, Dumaine A, Mathieu F, et al. A SNAP25 promoter variant is associated with early-onset bipolar disorder and a high expression level in brain [J]. Mol Psychiatry.2010; 15(7):748-755.
[43]Liu Y, Ye Z, Yang H, et al. Disturbances of soluble N-ethylmaleimide-sensitive factor attachment proteins in hippocampal synaptosomes contribute to cognitive impairment after repetitive formaldehyde inhalation in male rats [J]. Neuroscience.2010,169(3):1248-1254.
[44]Smith R, Klein P, Koc-Schmitz Y, et al. Loss of SNAP-25 and rabphilin 3 a in sensory-motor cortex in Huntington's disease [J]. J Neurochem.2007, 103(1):115-123.
[45]Katano T, Furue H, Okuda-Ashitaka E, et al. N-ethylmaleimide-sensitive fusion protein (NSF) is involved in central sensitization in the spinal cord through GluR2 subunit composition switch after inflammation [J]. Eur J Neurosci.2008; 27(12):3161-3170.
[46]Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence [J]. Physiol Rev.2001; 81(1):299-343.
[47]Li H, Zhou Y, Kang L, et al. Single and repeated morphine administrations differently regulate expression of N-ethylmaleimide-sensitive factor gene in the rat brain [J]. Neuroreport.2006,17(1):71-74.
[48]Yin S, Guan Z, Tang Y, et al. Abnormal expression of epilepsy-related gene ERG 1/NSF in the spontaneous recurrent seizure rats with spatial learning memory deficits induced by kainic acid [J]. Brain Res.2005; 1053(1-2):195-202.
[49]Castillo MA, Ghose S, Tamminga CA, et al. Deficits in syntaxin 1 phosphorylation in schizophrenia prefrontal cortex [J]. Biol. Psychiatry.2010,67(3): 208-216.
[50]Nakamura K, Anitha A, Yamada K, et al. Genetic and expression analyses reveal elevated expression of syntaxin 1A (STX1A) in high functioning autism [J]. Int J Neuropsychopharmacol.2008; 11(8):1073-1084.
[51]Eastwood SL, Harrison PJ. Hippocampal synaptic pathology in schizophrenia, bipolar disorder and major depression:a study of complexin mRNAs [J]. Mol Psychiatry.2000; 5(4):425-432.
[52]Begemann M, Grube S, Papiol S, et al. Modification of cognitive performance in schizophrenia by complexin 2 gene polymorphisms [J]. Arch. Gen. Psychiatry.2010,67(9):879-888.
[53]Dolly JO, O'Connell MA. Neurotherapeutics to inhibit exocytosis from sensory neurons for the control of chronic pain [J]. Curr Opin Pharmacol.2012; 12(1):100-108.
[54]Antonucci F, Rossi C, Gianfranceschi L, et al. Long-Distance Retrograde Effects of Botulinum Neurotoxin A [J]. J Neurosci.2008; 28(14):3689-3696.