Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts.
Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts.
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HEK293 细胞（一种人血脑屏障模型细胞）和 CCDS 患者来源的成纤维细胞中通过 SLC6A8（肌酸转运蛋白）转运环肌酸。
DOI：
10.1007/s11095-020-2779-0
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Ohtsuki S.
中科院分区：
文献类型：
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作者：
Uemura T;Ito S;Masuda T;Shimbo H;Goto T;Osaka H;Wada T;Couraud PO;Ohtsuki S.
目的环肌酸是一种肌酸类似物，是治疗由肌酸转运蛋白（CRT,SLC6A8）缺乏引起的脑肌酸缺乏综合征（CCDSs）的候选药物。本研究的目的是阐明高表达内源性CRT的HEK293细胞在人血脑屏障（BBB）模型hCMEC/D3细胞和ccds患者来源的CRT突变成纤维细胞中的环肌酸转运特征。方法将细胞与[14C]环肌酸（9 μM）和[14C]肌酸（9 μM）在37℃下孵育一定时间，有或没有抑制剂，同时通过脂质转染sirna。分别用靶向蛋白质组学和定量PCR法测定蛋白表达和mRNA表达。结果[14C]环肌酸被HEK293细胞吸收呈时间依赖性，并表现出饱和动力学。抑制和siRNA敲除研究表明，HEK293和hCMEC/D3细胞对[14C]环肌酸的摄取主要是由CRT和[14C]肌酸介导的。此外，发现ccds患者源性成纤维细胞对[14C]环肌酸和[14C]肌酸的摄取大大减少。结论本研究提示环肌酸是一个CRT底物，其中CRT是环肌酸流入脑血脑屏障的主要贡献者。我们的发现为使用环肌酸治疗ccds患者提供了重要的见解。
PurposeCyclocreatine, a creatine analog, is a candidate drug for treating patients with cerebral creatine deficiency syndromes (CCDSs) caused by creatine transporter (CRT,SLC6A8) deficiency, which reduces brain creatine level. The purpose of this study was to clarify the characteristics of cyclocreatine transport in HEK293 cells, which highly express endogenous CRT, in hCMEC/D3 cells, a human blood-brain barrier (BBB) model, and in CCDSs patient-derived fibroblasts with CRT mutations.MethodsCells were incubated at 37°C with [14C]cyclocreatine (9 μM) and [14C]creatine (9 μM) for specified periods of times in the presence or absence of inhibitors, while the siRNAs were transfected by lipofection. Protein expression and mRNA expression were quantified using targeted proteomics and quantitative PCR, respectively.Results[14C]Cyclocreatine was taken up by HEK293 cells in a time-dependent manner, while exhibiting saturable kinetics. The inhibition and siRNA knockdown studies demonstrated that the uptake of [14C]cyclocreatine by both HEK293 and hCMEC/D3 cells was mediated predominantly by CRT as well as [14C]creatine. In addition, uptake of [14C]cyclocreatine and [14C]creatine by the CCDSs patient-derived fibroblasts was found to be largely reduced.ConclusionThe present study suggests that cyclocreatine is a CRT substrate, where CRT is the predominant contributor to influx of cyclocreatine into the brain at the BBB. Our findings provide vital insights for the purposes of treating CCDSs patients using cyclocreatine.