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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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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.

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Abbreviations

ADP:

Adenosine diphosphate

AGAT:

L-Arginine: glycine amidinotransferase

ATP:

Adenosine triphosphate

BAP31:

B cell receptor-associated protein

BBB:

Blood-brain barrier

bFGF:

Basic fibroblast growth factor

CaMK2a:

Ca2+/calmodulin-dependent protein kinase a

CCDSs:

Cerebral creatine deficiency syndromes

CRT:

Creatine transporter

DMEM:

Dulbecco’s modified Eagle medium

FBS:

Fetal bovine serum

GAMT:

Guanidinoacetate methyltransferase

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GC:

Graphite carbon

GPA:

β-guanidinopropionic acid

HBSS:

Hank’s balanced salt solution

hCMEC:

Human cerebral microvascular endothelial cell

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

MRM:

Multiple reaction monitoring

PTS:

Phase-transfer surfactant

SDB:

Styrene-divinylbenzene

SLC6A8:

Solute carrier family 6 member 8

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ACKNOWLEDGMENTS AND DISCLOSURES

This study was supported in part by JSPS KAKENHI Grant Number JP26293035 and JP18H02590, and by AMED under Grant Number JP19ek0109396. Ohtsuki S is a full professor at Kumamoto University and is also a director of Proteomedix Frontiers. The other authors declare no competing interests.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan

    Tatsuki Uemura, Shingo Ito, Takeshi Masuda & Sumio Ohtsuki

  2. Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan

    Shingo Ito, Takeshi Masuda & Sumio Ohtsuki

  3. Division of Neurology, Kanagawa Children’s Medical Center, Kanagawa, 232-8555, Japan

    Hiroko Shimbo & Tomohide Goto

  4. Department of Pediatrics, Jichi Medical School, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan

    Hitoshi Osaka

  5. Department of Medical Ethics and Medical Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

    Takahito Wada

  6. Institut Cochin, Paris Descartes University, Inserm U1016, CNRS UMR8104, 75014, Paris, France

    Pierre-Olivier Couraud

Authors
  1. Tatsuki Uemura
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  2. Shingo Ito
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  9. Sumio Ohtsuki
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Contributions

All authors, Uemura T, Ito S, Masuda T, Shimbo H, Goto T, Osaka H, Wada T, Couraud PO, Ohtsuki S, contributed to study design and manuscript revision. Uemura T conducted the experiments and performed data analysis. Uemura T, Ito S. and Ohtsuki S wrote the manuscript. All the authors have provided their final approval for submission of this manuscript.

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Correspondence to Sumio Ohtsuki.

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Uemura, T., Ito, S., Masuda, T. et al. Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts. Pharm Res 37, 61 (2020). https://doi.org/10.1007/s11095-020-2779-0

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