The Functional Mechanism Of Sim2 Loacated At The Critical Region Of Down Syndrome

BACKGROUNDDown 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 hippocampusOBJECTIVETo construct the expression vector of mouse single-minded 2 (mSim2) and toexplore its effect on spatial memory and the potential mechanism.METHODSTotal 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.RESULTSAs 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.CONCLUSIONSim2 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 mSim2OBJECTIVETo 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 systemThe 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 MAD2L2The 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 proteinFunctions, 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 MAD2L2Two 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 MAD2L2MAD2L2 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.CONCLUSIONmSim2 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.