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ashed and then GA or PBS was added. Conditioned astrocyte medium was collected 18 h after drug withdrawal and 6 hours of washing. Media collected were spun briefly to obtain a cell-free conditioned medium that was used for treating isolated striatal neurons. Similar protocols were performed by pre-treating astrocytes with 20 mM FeTPPS, 2 mM FeTMPyP, 1 mM apocynin, or 20 mM U0126, during 30 to 60 min previous to add 5 mM GA. Statistical analysis Statistical analysis of data was performed with Sigma Stat 2.0 using student t-test or one-way ANOVA followed by Scheffe post hoc comparison if necessary. All results are presented as mean 6 SEM, p,0.05 was considered as significant. Acknowledgments We thank Dr. Alexandra Latini and Natalia Lago for critical reading of the manuscript, Pablo Diaz-Amarilla and Emiliano Trias for assistance in intraventricular injection and computational skills. Assessment of astrocyte mitochondrial potential, glutathione and oxidative levels in living astrocytes Mitochondrial potential was evaluated as previously described. Briefly, control and GA-treated astrocytes were incubated with the ratiometric dye JC1. The ratio Author Contributions Conceived and designed the experiments: SO-B LB AF MNS JCR GC MJ. Performed the experiments: SO-B AF MNS JCR GC MJ. Analyzed the data: SO-B LB AF MNS JCR GC MJ. Contributed reagents/materials/ analysis tools: SO-B LB AF GC. Wrote the paper: SO-B LB. 9 June 2011 | Volume 6 | Issue 6 | e20831 Astrocyte Damage and Striatal Degeneration 12. Funk CB, Prasad AN, Frosk P, Sauer S, Kolker S, et al. Neuropathological, biochemical and molecular findings in a glutaric acidemia type 1 cohort. Brain 128: 71122. 13. Zinnanti WJ, Lazovic J, Housman C, LaNoue K, O’Callaghan JP, et al. Mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type I. J Clin Invest 117: 3258270. 14. Sauer SW, Okun JG, Fricker G, Mahringer A, Muller I, et al. Intracerebral accumulation of glutaric and 3-hydroxyglutaric acids secondary to limited flux across the blood-brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl-CoA dehydrogenase deficiency. J Neurochem 97: 89910. 15. Hoffmann GF, Athanassopoulos S, Burlina AB, Duran M, de Klerk JB, et al. Clinical course, early diagnosis, treatment, and prevention of disease in glutaryl-CoA dehydrogenase deficiency. Neuropediatrics 27: 11523. 16. Leibel RL, Shih VE, Goodman SI, Bauman ML, McCabe ER, et al. Glutaric acidemia: a metabolic disorder causing progressive choreoathetosis. Neurology 30: 1163168. 17. Kolker S, Ahlemeyer B, Krieglstein J, Hoffmann GF Cerebral PCI32765 organic acid disorders induce neuronal damage via excitotoxic organic acids in vitro. Amino Acids 18: 310. 18. Soffer D, Amir N, Elpeleg ON, Gomori JM, Shalev RS, et al. Striatal degeneration and spongy myelinopathy in glutaric acidemia. J Neurol Sci 107: 19904. 19. Ferreira Gda C, Viegas CM, Schuck PF, Tonin A, Ribeiro CA, et al. Glutaric acid administration impairs energy metabolism in midbrain and skeletal muscle of young rats. Neurochem Res 30: 1123131. 20. Latini A, Ferreira GC, Scussiato K, Schuck PF, Solano AF, et al. Induction of oxidative stress by chronic and acute glutaric acid administration to rats. Cell Mol Neurobiol 27: 42338. 21. Lamp J, Keyser B, Koeller DM, Ullrich K, Braulke T, et al. Glutaric aciduria type 1 metabolites impair the succinate transport from astrocytic to neuronal cells. J Biol Chem. 22. Olivera S, Fernandez A, L

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