Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts
- 2 March 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Pharmaceutical Research
- Vol. 37 (3), 1-11
- https://doi.org/10.1007/s11095-020-2779-0
Abstract
Purpose Cyclocreatine, 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. Methods Cells 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. Conclusion The 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.Keywords
Funding Information
- Japan Society for the Promotion of Science (JP26293035, JP18H02590)
- AMED (JP19ek0109396)
This publication has 41 references indexed in Scilit:
- Cyclocreatine treatment improves cognition in mice with creatine transporter deficiencyJCI Insight, 2012
- Creatine Transporter (CrT; Slc6a8) Knockout Mice as a Model of Human CrT DeficiencyPLOS ONE, 2011
- Functional and immunocytochemical characterization of the creatine transporter in rat hippocampal neuronsJournal of Neurochemistry, 2010
- Phase Transfer Surfactant-Aided Trypsin Digestion for Membrane Proteome AnalysisJournal of Proteome Research, 2008
- X‐Linked creatine transporter deficiency in two patients with severe mental retardation and autismJournal of Inherited Metabolic Disease, 2006
- Creatine and guanidinoacetate: diagnostic markers for inborn errors in creatine biosynthesis and transportMolecular Genetics and Metabolism, 2004
- High Prevalence of SLC6A8 Deficiency in X-Linked Mental RetardationAmerican Journal of Human Genetics, 2004
- Irreversible brain creatine deficiency with elevated serum and urine creatine: A creatine transporter defect?Annals of Neurology, 2001
- FORMATION OF A SUPPLEMENTAL LONG TIME‐CONSTANT RESERVOIR OF HIGH ENERGY PHOSPHATE BY BRAIN IN VIVO AND IN VITRO AND ITS REVERSIBLE DEPLETION BY POTASSIUM DEPOLARIZATIONJournal of Neurochemistry, 1979