Improvements in an in-vitro assay for excitotoxicity by measurement of early gene (c-fos mRNA) levels.
Quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR) measurements were made to investigate the levels of c-fos mRNA as one measure of the expression of the c-fos gene. Exposure of mouse cerebellar granule cells to excitotoxic concentrations of glutamate (Glu) and aspartate (Asp) led to a changed time profile for mRNA expression, from a transient c-fos expression at 15-30 min to a delayed, elevated and sustained expression at later time points which was prevented by selective antagonism of the NMDA receptor but not of the AMPA/kainate receptor demonstrating that this c-fos induction was mediated through the specific activation of the NMDA Glu receptor subtype. The question as to whether c-fos expression changes could be used to predict excitotoxicity was addressed by testing the c-fos response of the cultures to several compounds, at low (and therefore non-toxic) and high (toxic) concentrations at two suitable time-points of exposure (30 and 240 min), in the presence and absence of Glu receptor antagonists. The compounds were divided into four groups, excitotoxins, neurotoxic but non-excitotoxic compounds, neuroactive but non-toxic compounds, and compounds that were toxic to other target organelles. The results of this study, using real-time RT-PCR, support the proposal that c-fos mRNA can be used as a specific biomarker of excitotoxicity and moreover encourage further studies to employ this highly sensitive, quantifiable and reproducible technique in a high throughput screen, with minimal use of animals for primary culture set-up. Furthermore, this test has the potential for application in screening newly-designed excitatory amino acid receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies.
Rogers
Annamarie
A
Bayer HealthCare, Pharma Research Centre, Aprather Weg, 42096, Wuppertal, Germany. Annamarie.Rogers@gmx.de
Schmuck
Gabriele
G
Scholz
Gabriele
G
Griffiths
Roger
R
Meredith
Clive
C
Schousboe
Arne
A
Campiani
Giuseppe
G
Williams
D Clive
DC
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay; D002470:Cell Survival; D002531:Cerebellum / Q000166:cytology; D018683:Excitatory Amino Acid Agents / Q000633:toxicity*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018846:Excitatory Amino Acids / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects*; D051379:Mice; D008815:Mice, Inbred Strains; D041681:NIH 3T3 Cells; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018683:Excitatory Amino Acid Agents; D018691:Excitatory Amino Acid Antagonists; D018846:Excitatory Amino Acids; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione