title: In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism.
abstract: BACKGROUND
  The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes.
  
  
  METHODS
  Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR.
  
  
  RESULTS
  D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells.
  
  
  CONCLUSIONS
  Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through de novo synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation.
authors.affiliation: Department of Experimental Medicine, La Sapienza Università di Roma, Viale del Policlinico 155, Rome 00161, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Carducci
authors.forename: Carla
authors.initials: C
authors.lastname: Carducci
authors.forename: Silvia
authors.initials: S
authors.lastname: Santagata
authors.forename: Enrico
authors.initials: E
authors.lastname: Adriano
authors.forename: Cristiana
authors.initials: C
authors.lastname: Artiola
authors.forename: Stefano
authors.initials: S
authors.lastname: Thellung
authors.forename: Elena
authors.initials: E
authors.lastname: Gatta
authors.forename: Mauro
authors.initials: M
authors.lastname: Robello
authors.forename: Tullio
authors.initials: T
authors.lastname: Florio
authors.forename: Italo
authors.initials: I
authors.lastname: Antonozzi
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Leuzzi
authors.forename: Maurizio
authors.initials: M
authors.lastname: Balestrino
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D003401:Creatine / Q000378:metabolism*; D050937:Guanidinoacetate N-Methyltransferase / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012436:S-Adenosylmethionine / Q000378:metabolism
chemical_list: D012436:S-Adenosylmethionine; D050937:Guanidinoacetate N-Methyltransferase; D003401:Creatine