唐氏综合征关键区段基因Sim2功能机制研究
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摘要
研究背景
唐氏综合征(Down Syndrome，DS)，亦称先天愚型，是人类最常见的染色体异常性疾病。尽管DS患者临床表现为脑、肌肉、心脏、骨骼和血液等多系统损伤，但以中枢神经系统表现最为突出——患者均有不同程度的智力发育迟缓和智力低下。DS患者大脑半球、额叶、颞叶和小脑体积均较正常人的小，且伴有神经元数量减少、树突棘数量减少和形态异常等结构变化。
研究表明，DS患者中枢神经系统的病变主要是由于21号染色体长臂2区2带(21q22)中少数几个基因的过量表达所致，21q22是引起DS表型的决定性部位。Sim2(single-minded 2)基因即位于该区域，提示Sim2可能在中枢神经系统发育和DS发生过程中发挥重要作用。最近研究表明，在人类胚胎发育期Sim2高表达于海马的锥体细胞和颗粒细胞层，以及小脑的外颗粒层和蒲肯野细胞层等，并且在大脑皮质中也有表达。这些部位在学习和记忆中起着重要作用，进一步提示Sim2基因参与了DS智力障碍的病理机制。然而，Sim2的功能机制尚未完全阐明。
本课题组前期研究发现，Sim2基因过表达可促进神经元凋亡，抑制神经元分化。为进一步探讨其作用的分子机制，本研究拟利用微注射法使小鼠Sim2基因(mSim2)在海马中高表达，观察其对大鼠空间记忆能力的影响，并检测synapsinⅠ的表达和磷酸化水平，以探讨其分子机制。此外，本研究还以mSim2作为诱饵基因，利用酵母双杂交方法筛查人胎脑cDNA文库，得到了与mSim2相互作用的候选基因，并进一步用激光共聚焦和免疫共沉淀方法加以证实，以期初步阐明Sim2的功能机制及其在DS中的致病机理。
第一部分过表达Sim2对大鼠空间记忆的影响及机制
目的：
构建mSim2真核表达载体，使其在大鼠海马局部过表达，观察其对大鼠空间记忆能力的影响并检测synapsinⅠ的表达和磷酸化水平，探讨其作用的分子机制。
方法：
(1)提取新生昆明小鼠脑组织总RNA，利用逆转录聚合酶链式反应(RT-PCR)，扩增出mSim2基因的开放阅读框，采用基因重组技术，将该基因定向克隆于真核表达载体pcDNA3.0中。
(2)选取成年SD大鼠48只，其中12只大鼠作为对照组，其余行水迷宫训练。训练5天后的大鼠分为3组：训练组、转染空载体组(转染pcDNA3.0)、转染mSim2组(转染pcDNA3-mSim2)。利用大鼠脑立体定位仪将载体注射于大鼠海马内，48小时后利用Morris水迷宫检测大鼠空间记忆能力，并利用RT-PCR和Western Blot方法检测mSim2在海马中的表达情况。
(3)利用RT-PCR和免疫组织化学方法检测synapsinⅠmRNA及蛋白表达。
结果：
(1)酶切鉴定和DNA测序结果表明，扩增的mSim2基因的开放阅读框正确地插入到真核表达载体pcDNA3.0中。RT-PCR和Westrern Blot结果显示，mSim2在转染pcDNA3-mSim2的大鼠海马中表达较其他各组明显增加，差异具有显著性(P＜0.05)。
(2)大鼠经Morris水迷宫训练5天后，寻找到平台的潜伏期为6.2±0.71s，转染mSim2组的大鼠寻找平台的潜伏期为14.2±1.22s，较训练组和转染空载体组明显延长，差异具有显著性(P＜0.05)。各组间大鼠的游泳速度无显著性差异。
(3)半定量RT-PCR结果表明，对照组大鼠海马synapsinⅠ表达水平较低。经水迷宫训练后，synapsinⅠmRNA表达显著增加。而在转染pcDNA3-mSim2组大鼠海马synapsinⅠ的表达较训练组和转染空载体组表达下降，差异具有显著性(图1-7，P＜0.05)。
(4)免疫组织化学结果显示，正常对照组大鼠synapsinⅠ在海马的CA1、CA3和齿状回区有痕量表达。经Morris水迷宫训练后，显微镜下见训练组和转染空载体组CA3区synapsinⅠ和磷酸化synapsinⅠ阳性反应增强，阳性细胞数目增多。图象分析结果表明，大鼠空间记忆过程促进海马CA3区synapsinⅠ蛋白表达和磷酸化水平，与未进行空间记忆大鼠(正常对照组)比较，差异具有显著性。而转染pcDNA3-mSim2组海马synapsinⅠ和磷酸化synapsinⅠ没有出现高表达现象，显微镜下见其阳性反应强度和阳性细胞数均弱于训练组和转染空载体组，图象分析结果显示差异具有显著性(P＜0.05)。
结论：
mSim2在大鼠海马中的过表达损害了大鼠空间记忆能力，抑制了synapsinⅠ的表达，提示mSim2可能通过影响突触信息传递在DS的病理机制中发挥重要作用。
第二部分mSim2相互作用蛋白的筛选和鉴定
目的：
采用酵母双杂交系统从人胎脑cDNA文库中筛选与mSim2相互作用的蛋白，并在细胞水平进行验证，为揭示Sim2的功能机制提供线索。
方法：
(1)酵母双杂交筛选mSim2相互作用蛋白
以第一部分构建的pcDNA3-mSim2为模板，PCR方法扩增用于酵母双杂交的目的片段，并构建于pGBKT7中，形成诱饵质粒pGBKT7-mSim2。利用醋酸锂转化法，将诱饵质粒转化酵母菌Y190，并涂布于相应的营养缺失培养板上进行毒性和自身激活检验。将人胎脑cDNA文库质粒和pGBKT7-mSim2共转化酵母菌，选择既能在相应的营养缺失培养板上生长，又具有活性的蓝色酵母克隆为阳性克隆。从阳性酵母中抽提质粒转化大肠杆菌，进行抗性筛选，再与诱饵质粒成对回复转染排除假阳性，扩增阳性质粒，进行测序和分析。
(2) mSim2相互作用蛋白的验证
分别构建带有pDsRed-monome-N1-mSim2和pEGFP-N1-MAD2L2真核表达载体，共转染PC12细胞。一方面，将转染的PC12细胞在双光子显微镜下观察mSim2和MAD2L2在细胞内是否存在共定位；另一方面，通过AgaroseA+G分别加入抗EGFP和抗Sim2的一抗进行免疫复合物共沉淀，再以抗Sim2和抗EGFP的抗体行Western blot检测，确定mSim2和MAD2L2是否存在相互作用。
(3) MAD2L2的生物信息学分析
利用在线分析软件和数据库对MAD2L2蛋白的结构特点、表达特征及其可能的相互作用蛋白网络进行生物信息学的初步分析预测。
结果：
(1)酵母双杂交筛选mSim2相互作用蛋白
构建的诱饵质粒pGBKT7-mSim2经酶切和测序表明重组克隆的重组方向及开放阅读框架均正确无误，并且诱饵载体对酵母无毒性，无自身激活报告基因的功能。以mSim2蛋白为“诱饵蛋白”，筛选人胎脑cDNA文库，共获得5个与mSim2诱饵蛋白相互作用的阳性克隆，其中3个克隆经测序证实均为MAD2L2。
(2) mSim2相互作用蛋白的验证
在双光子显微镜下观察到mSim2和MAD2L2存在共定位现象。免疫共沉淀结果显示抗EGFP沉淀MAD2L2蛋白的同时，mSim2也一同被沉淀。而用抗mSim2沉淀mSim2蛋白的同时，MAD2L2也一同被沉淀，这表明mSim2和MAD2L2在细胞水平存在相互作用。
(3) MAD2L2的生物信息学分析
MAD2L2蛋白序列特征表明，主要包括一个HORMA结构域。虚拟Northern杂交结果表明，在正常的中枢神经系统中表达水平很低，而在中枢肿瘤病变组织中表达水平上调。利用生物信息学对MAD2L2相互作用蛋白的初步分析发现其可能与细胞周期重要调控蛋白CDC20、ADAM17、ADAM9和PRCC等相互作用。
结论：
mSim2蛋白与MAD2L2蛋白存在相互作用，这为阐明mSim2的作用机制和揭示DS的发病机制提供了新的契机。
BACKGROUND
Down syndrome (DS) is the most common autosomal aneuploidy diseaseoccurring in 1.03 to 1.30 of 1000 livc births. The patients have many clinicalphenotypes, including craniofacial defects, deficiencies of the immune system, gutabnormalities, abnormalities of dermatoglyphics, hypotonia and cardiac defects.However, mental retardation occurs in all affected individuals, whereas othercharacteristics show variable penetrance. Total brain volume is consistently reducedin DS, with a disproportionately greater reduction in the cerebellum. This reduction isreadily apparent on post-mortem analysis, and has been measured quantitatively bymagnetic resonance imaging (MRI) studies reporting a total brain volume of 85% ofeuploid, and cerebellar volume further reduced to 73% of euploid.
Significant progresses have been made through careful correlation of cytogenetic,molecullar and clinical manifestations in individuals with translocations resulting intrisomy for a subset of human chromosome 21 genes (segmental trisomy 21). Mapscorrelating dosage imbalance of specific regions with specific characteristics provideduseful information about segments in which to search for the genes primarilyresponsible. A minimum region (21q22) is associated with many phenotypiccharacteristics of DS. Sim2 gene is located in this region. Sim2 showed differentialexpression in pyramidal and granular cell layers of hippocampal formation, in corticalcells and in cerebellar external granular and Purkinje cell layers. Sim2 expression inembryonic and fetal brain could suggest a potential role in human central nervoussystem (CNS) development, in agreement with Drosophila and mouse Sim mutantphenotypes and with the conservation of the Sim function in CNS development fromdrosophila to Human. However, the mechanism of Sim2 is still unelucidated.
We previously in vitro study found that mSim2 transfection increased neuronapoptosis and inhibited its differentiation under the fund of National Natural ScienceFoundation of China. To further explore the pathogeneic mechanism of Sim2, we planto overexpress Sim2 in the hippocampus to study its effect on space memory and toscreen its interactiong proteins with yeast two-hybrid. Then co-localization and co-immunoprecipitation were used to verify the interaction of Sim2 with the targetproteins. Through this study, we hope to eluciate the functional mechanism of Sim2and explore the pathophysiology of DS.
PARTⅠEffects of overexpression of Sim2 on spatial memory and expressionof synapsin I in rat hippocampus
OBJECTIVE
To construct the expression vector of mouse single-minded 2 (mSim2) and toexplore its effect on spatial memory and the potential mechanism.
METHODS
Total RNA was obtained from a fetal male Kunming mouse. RT-PCR was used toamplify the open reading frarne of mouse mSim2. Then ORF of mSim2 wasconstructed into pcDNA3.0 plasmid, pcDNA3.0-mSim2 plasmid wrapped withliposome was bilaterally injected into the hippocampus of rats. The expression ofmSim2 was detected by RT-PCR and Western Blot. The effect of overexpressingmSim2 on spatial memory was detected by Morris water maze task. The expression ofsynapsin I was detected by RT-PCR and immunohistochemistry, respectively. Thephosphosynapsin was also examined by immunohistochemistry.
RESULTS
As demonstrated by RT-PCR and Western Blot, mSim2 was successfullyoverexpressed in the hippocampus of rats, and pcDNA3/mSim2-transfected ratsshowed longer latency to find the hidden platform compared withpcDNA3-transfected rats (P＜0.05). Synapsin I mRNA and protein expression weredecreased significantly by mSim2 transfection, as demonstrated by RT-PCR andimmunohistochemistry (P＜0.05). Moreover, the expression profile ofphosphosynapsin was similar to that of synapsin I.
CONCLUSION
Sim2 could impair the ability of learning and memory by inhibiting synaptictransmission, and may play a crucial role in the pathogenesis of DS.
PARTⅡScreening and verifying the interacting proteins of mSim2
OBJECTIVE
To screen and identification of the potential interacting proteins with mSim2,and perform related bioinformatics analysis to predict the potential fuctionalmechanism.
METHODS
(1) Screening of mSim2 interaction proteins with yeast two-hybrid system
The fragment of mSim2 was amplified with PCR and then cloned into thepGBKT7 to form the bait plasmid pGBKT7- mSim2. The constructed plasmid wastransformed into the Y190 yeast with LiAc. The toxicity and activity were detected.The human fetal brain cDNA library and pGBKT7- mSim2 were co-transformed intothe Y190 yeast. If the transformed Y190 clones grew on the minus culture plate andwere blue, they were regarded as putative positive clones. The plasmid of thepositive clones were then extracted and used to verify the interaction and exclude thefalse positive clones. The the plasmids were sequenced and analyzed.
(2) Verification the interaction of mSim2 and MAD2L2
The plasmids of pDsRed-monomer -N1-mSim2 and pEGFP-N1-MAD2L2 wereconstructed and co-transfected into PC12 cells. Interaction of mSim2 and MAD2L2was identified by co-localization analysis and co-immunoprecipation assay. Thetransfected cellls were lysed, and proteins were purified by anti-EGFP or anti-Sim2respectively. The proteins were analyzed by Western blot with anti-Sim2 oranti-EGFP respectively. The transfected cells were also observed with the twophoton laser scanning system.
(3) Bioinformatical analysis of MAD2L2 protein
Functions, expression patters and possible interaction networks of MAD2L2protein were predicted by several kinds of bioinformatics and online databases.
RESULTS
(1) Screening of mSim2-interacting proteins in yeast two-hybrid system
Five positive clones in yeast two-hybrid screening systems were found afterhuman fetal brain cDNA library had been screened. Three of five selected clones weresequenced and confirmed as gene MAD2L2. MAD2L2 was regarded as a candidatemSim2-interaeting protein to be identified.
(2) Verification the interaction of mSim2 with MAD2L2
Two eukaryotic expressing vectors of pDsRed-monomer-N1-mSim2 andpEGFP-N1-MAD2L2 were constructed successfully. When they were co-tranfectedinto PC12 cell, MAD2L2 and mSim2 were co-localized in cellular nucleus.Consistent with co-localization result, mSim2 was co-immunoprecipitated withMAD2L2.
(3) Bioinformaties analysis of the potential function of MAD2L2
MAD2L2 has one conserved HORMA domain. The results of digital Northern blotsshowed that MAD2L2 was expressed in the spinal cord, cerebellum and cortex in thenormal central nervous system. When cancers occurred in the nervous system, theexpression of MAD2L2 increased. MAD2L2 can interact with proteins important tocell cycle regulation, which suggests that mSim2-MAD2L2 may be involved inneuron apoptosis and differentiation.
CONCLUSION
mSim2 can interact with MAD2L2, which is a new mSim2 interacting protein. Thepresent research work is important to explore the functional mechanism of Sim2 andto elucidate the pathophysiology of DS.
唐氏综合征(Down Syndrome，DS)，亦称先天愚型，是人类最常见的染色体异常性疾病。尽管DS患者临床表现为脑、肌肉、心脏、骨骼和血液等多系统损伤，但以中枢神经系统表现最为突出——患者均有不同程度的智力发育迟缓和智力低下。DS患者大脑半球、额叶、颞叶和小脑体积均较正常人的小，且伴有神经元数量减少、树突棘数量减少和形态异常等结构变化。
研究表明，DS患者中枢神经系统的病变主要是由于21号染色体长臂2区2带(21q22)中少数几个基因的过量表达所致，21q22是引起DS表型的决定性部位。Sim2(single-minded 2)基因即位于该区域，提示Sim2可能在中枢神经系统发育和DS发生过程中发挥重要作用。最近研究表明，在人类胚胎发育期Sim2高表达于海马的锥体细胞和颗粒细胞层，以及小脑的外颗粒层和蒲肯野细胞层等，并且在大脑皮质中也有表达。这些部位在学习和记忆中起着重要作用，进一步提示Sim2基因参与了DS智力障碍的病理机制。然而，Sim2的功能机制尚未完全阐明。
本课题组前期研究发现，Sim2基因过表达可促进神经元凋亡，抑制神经元分化。为进一步探讨其作用的分子机制，本研究拟利用微注射法使小鼠Sim2基因(mSim2)在海马中高表达，观察其对大鼠空间记忆能力的影响，并检测synapsinⅠ的表达和磷酸化水平，以探讨其分子机制。此外，本研究还以mSim2作为诱饵基因，利用酵母双杂交方法筛查人胎脑cDNA文库，得到了与mSim2相互作用的候选基因，并进一步用激光共聚焦和免疫共沉淀方法加以证实，以期初步阐明Sim2的功能机制及其在DS中的致病机理。
第一部分过表达Sim2对大鼠空间记忆的影响及机制
目的：
构建mSim2真核表达载体，使其在大鼠海马局部过表达，观察其对大鼠空间记忆能力的影响并检测synapsinⅠ的表达和磷酸化水平，探讨其作用的分子机制。
方法：
(1)提取新生昆明小鼠脑组织总RNA，利用逆转录聚合酶链式反应(RT-PCR)，扩增出mSim2基因的开放阅读框，采用基因重组技术，将该基因定向克隆于真核表达载体pcDNA3.0中。
(2)选取成年SD大鼠48只，其中12只大鼠作为对照组，其余行水迷宫训练。训练5天后的大鼠分为3组：训练组、转染空载体组(转染pcDNA3.0)、转染mSim2组(转染pcDNA3-mSim2)。利用大鼠脑立体定位仪将载体注射于大鼠海马内，48小时后利用Morris水迷宫检测大鼠空间记忆能力，并利用RT-PCR和Western Blot方法检测mSim2在海马中的表达情况。
(3)利用RT-PCR和免疫组织化学方法检测synapsinⅠmRNA及蛋白表达。
结果：
(1)酶切鉴定和DNA测序结果表明，扩增的mSim2基因的开放阅读框正确地插入到真核表达载体pcDNA3.0中。RT-PCR和Westrern Blot结果显示，mSim2在转染pcDNA3-mSim2的大鼠海马中表达较其他各组明显增加，差异具有显著性(P＜0.05)。
(2)大鼠经Morris水迷宫训练5天后，寻找到平台的潜伏期为6.2±0.71s，转染mSim2组的大鼠寻找平台的潜伏期为14.2±1.22s，较训练组和转染空载体组明显延长，差异具有显著性(P＜0.05)。各组间大鼠的游泳速度无显著性差异。
(3)半定量RT-PCR结果表明，对照组大鼠海马synapsinⅠ表达水平较低。经水迷宫训练后，synapsinⅠmRNA表达显著增加。而在转染pcDNA3-mSim2组大鼠海马synapsinⅠ的表达较训练组和转染空载体组表达下降，差异具有显著性(图1-7，P＜0.05)。
(4)免疫组织化学结果显示，正常对照组大鼠synapsinⅠ在海马的CA1、CA3和齿状回区有痕量表达。经Morris水迷宫训练后，显微镜下见训练组和转染空载体组CA3区synapsinⅠ和磷酸化synapsinⅠ阳性反应增强，阳性细胞数目增多。图象分析结果表明，大鼠空间记忆过程促进海马CA3区synapsinⅠ蛋白表达和磷酸化水平，与未进行空间记忆大鼠(正常对照组)比较，差异具有显著性。而转染pcDNA3-mSim2组海马synapsinⅠ和磷酸化synapsinⅠ没有出现高表达现象，显微镜下见其阳性反应强度和阳性细胞数均弱于训练组和转染空载体组，图象分析结果显示差异具有显著性(P＜0.05)。
结论：
mSim2在大鼠海马中的过表达损害了大鼠空间记忆能力，抑制了synapsinⅠ的表达，提示mSim2可能通过影响突触信息传递在DS的病理机制中发挥重要作用。
第二部分mSim2相互作用蛋白的筛选和鉴定
目的：
采用酵母双杂交系统从人胎脑cDNA文库中筛选与mSim2相互作用的蛋白，并在细胞水平进行验证，为揭示Sim2的功能机制提供线索。
方法：
(1)酵母双杂交筛选mSim2相互作用蛋白
以第一部分构建的pcDNA3-mSim2为模板，PCR方法扩增用于酵母双杂交的目的片段，并构建于pGBKT7中，形成诱饵质粒pGBKT7-mSim2。利用醋酸锂转化法，将诱饵质粒转化酵母菌Y190，并涂布于相应的营养缺失培养板上进行毒性和自身激活检验。将人胎脑cDNA文库质粒和pGBKT7-mSim2共转化酵母菌，选择既能在相应的营养缺失培养板上生长，又具有活性的蓝色酵母克隆为阳性克隆。从阳性酵母中抽提质粒转化大肠杆菌，进行抗性筛选，再与诱饵质粒成对回复转染排除假阳性，扩增阳性质粒，进行测序和分析。
(2) mSim2相互作用蛋白的验证
分别构建带有pDsRed-monome-N1-mSim2和pEGFP-N1-MAD2L2真核表达载体，共转染PC12细胞。一方面，将转染的PC12细胞在双光子显微镜下观察mSim2和MAD2L2在细胞内是否存在共定位；另一方面，通过AgaroseA+G分别加入抗EGFP和抗Sim2的一抗进行免疫复合物共沉淀，再以抗Sim2和抗EGFP的抗体行Western blot检测，确定mSim2和MAD2L2是否存在相互作用。
(3) MAD2L2的生物信息学分析
利用在线分析软件和数据库对MAD2L2蛋白的结构特点、表达特征及其可能的相互作用蛋白网络进行生物信息学的初步分析预测。
结果：
(1)酵母双杂交筛选mSim2相互作用蛋白
构建的诱饵质粒pGBKT7-mSim2经酶切和测序表明重组克隆的重组方向及开放阅读框架均正确无误，并且诱饵载体对酵母无毒性，无自身激活报告基因的功能。以mSim2蛋白为“诱饵蛋白”，筛选人胎脑cDNA文库，共获得5个与mSim2诱饵蛋白相互作用的阳性克隆，其中3个克隆经测序证实均为MAD2L2。
(2) mSim2相互作用蛋白的验证
在双光子显微镜下观察到mSim2和MAD2L2存在共定位现象。免疫共沉淀结果显示抗EGFP沉淀MAD2L2蛋白的同时，mSim2也一同被沉淀。而用抗mSim2沉淀mSim2蛋白的同时，MAD2L2也一同被沉淀，这表明mSim2和MAD2L2在细胞水平存在相互作用。
(3) MAD2L2的生物信息学分析
MAD2L2蛋白序列特征表明，主要包括一个HORMA结构域。虚拟Northern杂交结果表明，在正常的中枢神经系统中表达水平很低，而在中枢肿瘤病变组织中表达水平上调。利用生物信息学对MAD2L2相互作用蛋白的初步分析发现其可能与细胞周期重要调控蛋白CDC20、ADAM17、ADAM9和PRCC等相互作用。
结论：
mSim2蛋白与MAD2L2蛋白存在相互作用，这为阐明mSim2的作用机制和揭示DS的发病机制提供了新的契机。
BACKGROUND
Down syndrome (DS) is the most common autosomal aneuploidy diseaseoccurring in 1.03 to 1.30 of 1000 livc births. The patients have many clinicalphenotypes, including craniofacial defects, deficiencies of the immune system, gutabnormalities, abnormalities of dermatoglyphics, hypotonia and cardiac defects.However, mental retardation occurs in all affected individuals, whereas othercharacteristics show variable penetrance. Total brain volume is consistently reducedin DS, with a disproportionately greater reduction in the cerebellum. This reduction isreadily apparent on post-mortem analysis, and has been measured quantitatively bymagnetic resonance imaging (MRI) studies reporting a total brain volume of 85% ofeuploid, and cerebellar volume further reduced to 73% of euploid.
Significant progresses have been made through careful correlation of cytogenetic,molecullar and clinical manifestations in individuals with translocations resulting intrisomy for a subset of human chromosome 21 genes (segmental trisomy 21). Mapscorrelating dosage imbalance of specific regions with specific characteristics provideduseful information about segments in which to search for the genes primarilyresponsible. A minimum region (21q22) is associated with many phenotypiccharacteristics of DS. Sim2 gene is located in this region. Sim2 showed differentialexpression in pyramidal and granular cell layers of hippocampal formation, in corticalcells and in cerebellar external granular and Purkinje cell layers. Sim2 expression inembryonic and fetal brain could suggest a potential role in human central nervoussystem (CNS) development, in agreement with Drosophila and mouse Sim mutantphenotypes and with the conservation of the Sim function in CNS development fromdrosophila to Human. However, the mechanism of Sim2 is still unelucidated.
We previously in vitro study found that mSim2 transfection increased neuronapoptosis and inhibited its differentiation under the fund of National Natural ScienceFoundation of China. To further explore the pathogeneic mechanism of Sim2, we planto overexpress Sim2 in the hippocampus to study its effect on space memory and toscreen its interactiong proteins with yeast two-hybrid. Then co-localization and co-immunoprecipitation were used to verify the interaction of Sim2 with the targetproteins. Through this study, we hope to eluciate the functional mechanism of Sim2and explore the pathophysiology of DS.
PARTⅠEffects of overexpression of Sim2 on spatial memory and expressionof synapsin I in rat hippocampus
OBJECTIVE
To construct the expression vector of mouse single-minded 2 (mSim2) and toexplore its effect on spatial memory and the potential mechanism.
METHODS
Total RNA was obtained from a fetal male Kunming mouse. RT-PCR was used toamplify the open reading frarne of mouse mSim2. Then ORF of mSim2 wasconstructed into pcDNA3.0 plasmid, pcDNA3.0-mSim2 plasmid wrapped withliposome was bilaterally injected into the hippocampus of rats. The expression ofmSim2 was detected by RT-PCR and Western Blot. The effect of overexpressingmSim2 on spatial memory was detected by Morris water maze task. The expression ofsynapsin I was detected by RT-PCR and immunohistochemistry, respectively. Thephosphosynapsin was also examined by immunohistochemistry.
RESULTS
As demonstrated by RT-PCR and Western Blot, mSim2 was successfullyoverexpressed in the hippocampus of rats, and pcDNA3/mSim2-transfected ratsshowed longer latency to find the hidden platform compared withpcDNA3-transfected rats (P＜0.05). Synapsin I mRNA and protein expression weredecreased significantly by mSim2 transfection, as demonstrated by RT-PCR andimmunohistochemistry (P＜0.05). Moreover, the expression profile ofphosphosynapsin was similar to that of synapsin I.
CONCLUSION
Sim2 could impair the ability of learning and memory by inhibiting synaptictransmission, and may play a crucial role in the pathogenesis of DS.
PARTⅡScreening and verifying the interacting proteins of mSim2
OBJECTIVE
To screen and identification of the potential interacting proteins with mSim2,and perform related bioinformatics analysis to predict the potential fuctionalmechanism.
METHODS
(1) Screening of mSim2 interaction proteins with yeast two-hybrid system
The fragment of mSim2 was amplified with PCR and then cloned into thepGBKT7 to form the bait plasmid pGBKT7- mSim2. The constructed plasmid wastransformed into the Y190 yeast with LiAc. The toxicity and activity were detected.The human fetal brain cDNA library and pGBKT7- mSim2 were co-transformed intothe Y190 yeast. If the transformed Y190 clones grew on the minus culture plate andwere blue, they were regarded as putative positive clones. The plasmid of thepositive clones were then extracted and used to verify the interaction and exclude thefalse positive clones. The the plasmids were sequenced and analyzed.
(2) Verification the interaction of mSim2 and MAD2L2
The plasmids of pDsRed-monomer -N1-mSim2 and pEGFP-N1-MAD2L2 wereconstructed and co-transfected into PC12 cells. Interaction of mSim2 and MAD2L2was identified by co-localization analysis and co-immunoprecipation assay. Thetransfected cellls were lysed, and proteins were purified by anti-EGFP or anti-Sim2respectively. The proteins were analyzed by Western blot with anti-Sim2 oranti-EGFP respectively. The transfected cells were also observed with the twophoton laser scanning system.
(3) Bioinformatical analysis of MAD2L2 protein
Functions, expression patters and possible interaction networks of MAD2L2protein were predicted by several kinds of bioinformatics and online databases.
RESULTS
(1) Screening of mSim2-interacting proteins in yeast two-hybrid system
Five positive clones in yeast two-hybrid screening systems were found afterhuman fetal brain cDNA library had been screened. Three of five selected clones weresequenced and confirmed as gene MAD2L2. MAD2L2 was regarded as a candidatemSim2-interaeting protein to be identified.
(2) Verification the interaction of mSim2 with MAD2L2
Two eukaryotic expressing vectors of pDsRed-monomer-N1-mSim2 andpEGFP-N1-MAD2L2 were constructed successfully. When they were co-tranfectedinto PC12 cell, MAD2L2 and mSim2 were co-localized in cellular nucleus.Consistent with co-localization result, mSim2 was co-immunoprecipitated withMAD2L2.
(3) Bioinformaties analysis of the potential function of MAD2L2
MAD2L2 has one conserved HORMA domain. The results of digital Northern blotsshowed that MAD2L2 was expressed in the spinal cord, cerebellum and cortex in thenormal central nervous system. When cancers occurred in the nervous system, theexpression of MAD2L2 increased. MAD2L2 can interact with proteins important tocell cycle regulation, which suggests that mSim2-MAD2L2 may be involved inneuron apoptosis and differentiation.
CONCLUSION
mSim2 can interact with MAD2L2, which is a new mSim2 interacting protein. Thepresent research work is important to explore the functional mechanism of Sim2 andto elucidate the pathophysiology of DS.
引文
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3. Yamaki, A., Noda, S., Kudoh, J., et al. The mammalian single-minded (SIM) gene: mouse cDNA structure and diencephalic expression indicate a candidate gene for Down's syndrome. Genomics, 1996, 35:136-143.
4. Rachidi M, Lopes C, Charron G, et al. Spatial and temporal localization during embryonic and fetal human development of the transcription factor SIM2 in brain regions altered in Down syndrome.Int J Dev Neurosci, 2005,23:475-484.
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6. Ema M, Suzuki M, Morita M, Hiros K, et al. (1996) cDNA Cloning of a Murine Homologue of Drosophila Single-Minded, Its mRNA Expression in Mouse Development, and Chromosome Localization. Biochemical and Biophysical Research Communications 218, 588-594 .
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3. Yamaki, A., Noda, S., Kudoh, J., et al. The mammalian single-minded (SIM) gene: mouse cDNA structure and diencephalic expression indicate a candidate gene for Down's syndrome. Genomics, 1996, 35:136-143.
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6. Ema M, Suzuki M, Morita M, Hiros K, et al. (1996) cDNA Cloning of a Murine Homologue of Drosophila Single-Minded, Its mRNA Expression in Mouse Development, and Chromosome Localization. Biochemical and Biophysical Research Communications 218, 588-594 .
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