OBJECTIVE Selective serotonin reuptake inhibitors(SSRIs) bind 5-HT transporters,leading to the accumulation of 5-HT and amelioration of depression.Although different mouse strain showed different sensitivity to SSRIs ...OBJECTIVE Selective serotonin reuptake inhibitors(SSRIs) bind 5-HT transporters,leading to the accumulation of 5-HT and amelioration of depression.Although different mouse strain showed different sensitivity to SSRIs in mouse models of depression,the reason for these strain differences remains unclear.Here,therefore,in the present study,we examined immobility time and locomotor activity in two mouse strains,namely,C57BL/6 J and DBA/2 J mice,and the effects of the SSRIs fluoxetine.Furthermore,we analyzed 5-HT transporter binding and reuptake inhibition in both strains to explore their relationship with the immobility and locomotor activity effects of the three SSRIs in these two mouse strains.METHODS Strain differences in SSRI effects in the tail suspension test(TST) and forced swimming test(FST).To initiate our studies,we sought to confirm that SERT strain variation did not alter SERT protein expression,5-HT recognition,or uptake activity when expressed in C57BL/6 J and DBA/2 J mice.Radioligand binding assays were conducted to determine the affinity of the SSRIs for the 5-HT transporters in the two mouse strains.RESULTS SSRI citalopram dose-dependently reduced immobility time in both the FST and TST in DBA/2 J but not C57BL/6 J mouse strains,whereas fluoxetine showed opposite results.Paroxetine reduced immobility time similarly in both strains.The affinity of citalopram for the 5-HT transporter in DBA/2 J mice was 700-fold higher than that for in C57BL/6 J mice,whereas the affinity of fluoxetine in C57BL/6 J mice was 100-fold higher than that in the DBA/2 J mouse.Furthermore,High citalopram concentrations were required to [3 H]5-HT uptake in C57BL/6 J but not DBA/2 J mouse cortical synaptosomes,whereas fluoxetine also showed opposite results.CONCLUSION Immobility duration depends on 5-HT transporter binding levels,leading to apparent strain differences in immobility time in FST and TST.Furthermore,differences in 5-HT transporter binding may cause variations in SSRI responses on behaviors.SERT mutation mice maintained sensitivity to paroxetine,an antidepressant that is unaffected by the mouse mutation.Therefore,the background strain of these mice likely contributes to the acute behavioral actions of SSRIs in immobility time.These differences may help to explain some of the discrepancies in studies that used these strains of mice to examine the role of 5-HT in mouse models of depression.Future studies should investigate additional neural substrates and molecular mechanisms underlying strain variations in mouse models of depression to help identify genetic predispositions to this disorder in humans.展开更多
目的·构建5-羟色胺化(serotonylation,5-HT化)修饰蛋白研究体系,为寻找和发现5-HT化修饰蛋白提供方法学基础。方法·基于癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和基因型-组织表达(Genotype-Tissue Expression,GT...目的·构建5-羟色胺化(serotonylation,5-HT化)修饰蛋白研究体系,为寻找和发现5-HT化修饰蛋白提供方法学基础。方法·基于癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和基因型-组织表达(Genotype-Tissue Expression,GTEx)数据库分析编码5-HT化修饰的关键酶的基因转谷氨酰胺酶2(transglutaminase 2,TGM2)和编码5-羟色胺转运体(serotonin transporter,SERT)的基因溶质载体家族6(solute carrier family 6,SLC6A4)在正常生理组织和肿瘤组织中的表达情况。利用5-羟色胺盐酸盐分步合成5-羟色胺衍生物5-PT(5-propargyltryptamide),并利用傅里叶变换红外光谱(FT-IR)、核磁共振氢谱1H-NMR、核磁共振碳谱13C-NMR和飞行时间质谱(TOF-MS)等手段进行分析和结构表征。通过流式细胞术检测5-PT在人胰腺癌细胞AsPC-1和小鼠免疫细胞[包括CD4^(+)T细胞、CD8^(+)T细胞以及髓系巨噬细胞(bone marrow derived macrophage,BMDM)]的胞内摄入情况。通过点击化学反应、免疫共沉淀以及质谱分析技术寻找和鉴定发生5-HT化修饰的蛋白,并进行京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。结果·生物信息学相关分析显示TGM2和SLC6A4在生物体内正常组织和肿瘤组织广泛存在。流式细胞术结果显示合成的5-PT可通过细胞表面的SERT摄取进入人胰腺癌细胞AsPC-1和小鼠免疫细胞(包括CD4^(+)T细胞、CD8^(+)T细胞以及BMDM)。质谱分析数据显示,在各个细胞蛋白的5-PT处理组都富集到了丰富的5-HT化修饰蛋白。KEGG富集分析显示这些蛋白参与糖酵解和氨基酸合成相关通路。结论·利用合成的5-PT成功构建了5-HT化修饰蛋白的研究体系,在不同的细胞中富集得到多个5-HT化修饰的蛋白,为研究蛋白的5-HT化修饰及其功能提供了相对简单高效的研究手段。展开更多
文摘OBJECTIVE Selective serotonin reuptake inhibitors(SSRIs) bind 5-HT transporters,leading to the accumulation of 5-HT and amelioration of depression.Although different mouse strain showed different sensitivity to SSRIs in mouse models of depression,the reason for these strain differences remains unclear.Here,therefore,in the present study,we examined immobility time and locomotor activity in two mouse strains,namely,C57BL/6 J and DBA/2 J mice,and the effects of the SSRIs fluoxetine.Furthermore,we analyzed 5-HT transporter binding and reuptake inhibition in both strains to explore their relationship with the immobility and locomotor activity effects of the three SSRIs in these two mouse strains.METHODS Strain differences in SSRI effects in the tail suspension test(TST) and forced swimming test(FST).To initiate our studies,we sought to confirm that SERT strain variation did not alter SERT protein expression,5-HT recognition,or uptake activity when expressed in C57BL/6 J and DBA/2 J mice.Radioligand binding assays were conducted to determine the affinity of the SSRIs for the 5-HT transporters in the two mouse strains.RESULTS SSRI citalopram dose-dependently reduced immobility time in both the FST and TST in DBA/2 J but not C57BL/6 J mouse strains,whereas fluoxetine showed opposite results.Paroxetine reduced immobility time similarly in both strains.The affinity of citalopram for the 5-HT transporter in DBA/2 J mice was 700-fold higher than that for in C57BL/6 J mice,whereas the affinity of fluoxetine in C57BL/6 J mice was 100-fold higher than that in the DBA/2 J mouse.Furthermore,High citalopram concentrations were required to [3 H]5-HT uptake in C57BL/6 J but not DBA/2 J mouse cortical synaptosomes,whereas fluoxetine also showed opposite results.CONCLUSION Immobility duration depends on 5-HT transporter binding levels,leading to apparent strain differences in immobility time in FST and TST.Furthermore,differences in 5-HT transporter binding may cause variations in SSRI responses on behaviors.SERT mutation mice maintained sensitivity to paroxetine,an antidepressant that is unaffected by the mouse mutation.Therefore,the background strain of these mice likely contributes to the acute behavioral actions of SSRIs in immobility time.These differences may help to explain some of the discrepancies in studies that used these strains of mice to examine the role of 5-HT in mouse models of depression.Future studies should investigate additional neural substrates and molecular mechanisms underlying strain variations in mouse models of depression to help identify genetic predispositions to this disorder in humans.
文摘目的·构建5-羟色胺化(serotonylation,5-HT化)修饰蛋白研究体系,为寻找和发现5-HT化修饰蛋白提供方法学基础。方法·基于癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和基因型-组织表达(Genotype-Tissue Expression,GTEx)数据库分析编码5-HT化修饰的关键酶的基因转谷氨酰胺酶2(transglutaminase 2,TGM2)和编码5-羟色胺转运体(serotonin transporter,SERT)的基因溶质载体家族6(solute carrier family 6,SLC6A4)在正常生理组织和肿瘤组织中的表达情况。利用5-羟色胺盐酸盐分步合成5-羟色胺衍生物5-PT(5-propargyltryptamide),并利用傅里叶变换红外光谱(FT-IR)、核磁共振氢谱1H-NMR、核磁共振碳谱13C-NMR和飞行时间质谱(TOF-MS)等手段进行分析和结构表征。通过流式细胞术检测5-PT在人胰腺癌细胞AsPC-1和小鼠免疫细胞[包括CD4^(+)T细胞、CD8^(+)T细胞以及髓系巨噬细胞(bone marrow derived macrophage,BMDM)]的胞内摄入情况。通过点击化学反应、免疫共沉淀以及质谱分析技术寻找和鉴定发生5-HT化修饰的蛋白,并进行京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。结果·生物信息学相关分析显示TGM2和SLC6A4在生物体内正常组织和肿瘤组织广泛存在。流式细胞术结果显示合成的5-PT可通过细胞表面的SERT摄取进入人胰腺癌细胞AsPC-1和小鼠免疫细胞(包括CD4^(+)T细胞、CD8^(+)T细胞以及BMDM)。质谱分析数据显示,在各个细胞蛋白的5-PT处理组都富集到了丰富的5-HT化修饰蛋白。KEGG富集分析显示这些蛋白参与糖酵解和氨基酸合成相关通路。结论·利用合成的5-PT成功构建了5-HT化修饰蛋白的研究体系,在不同的细胞中富集得到多个5-HT化修饰的蛋白,为研究蛋白的5-HT化修饰及其功能提供了相对简单高效的研究手段。
