Monium are produced by brain cells under the action of GA and 3-OHGA, suggesting a central liberation of ammonium in GA-I. Following the guidelines for GA-I, ammonium is not routinely determined during an acute illness [10,11], but could be worth to be measured in CSF. Ammonium is known to be toxic for brain cells causing reduced axonal elongation [16] as well as neuronal and oligodendrocytic cell death [15,18], which correlates with the brain atrophy and white matter changes observed in patients with primary hyperammonemias [20]. Its detection in brain cell cultures challenged with GA and 3-OHGA immediately raises the question of a potential role for ammonium in brain damage occurring in GA-I patients. As urea cycle is not active in central nervous system, ammonium produced during amino acid catabolism is mainly detoxified through amination of glutamate to Title Loaded From File glutamine by the enzyme glutamine synthetase. This enzyme is exclusivelyBrain Cell Damage in Glutaric Title Loaded From File Aciduria Type IFigure 5. Effects of GA and 3-OHGA on biochemical parameters measured in culture medium. Glucose (A), lactate (B), ammonium (C) and glutamine (D) were measured in the medium of cultures treated with protocols A (DIV 8) or B (DIV 14). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; *p,0.05, **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type IFigure 6. Evaluation of cell death after treatment with GA and 3-OHGA. (A; left panel) Immunohistochemical staining for cleaved caspase-3 (red signal). Scale bar: 100 mm. (A; right panel) Representative western blots with data quantification of whole-cell lysates for full length caspase-3 and the large fragment of cleaved (e.g. activated) caspase-3 for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of cleaved caspase-3 are expressed as percentage of respective controls. The values represent the mean 6 SEM from 3 replicates taken from 2 independent experiments. (B) In situ cell death assay with TUNEL (green signal) and cleaved caspase-3 (red signal) on DIV 8 (protocol A). Merge of both signals leads to double-stained cells appearing in yellow. Scale bar: 100 mm. (C) LDH in culture medium of cultures from protocol A (DIV 8, above) and protocol B (DIV 14, below). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type Isupported by the observation of neuronal loss in the Gcdh2/2 mouse model [13]. Analysis of media from treated and control cultures on DIV 14 showed a marked increase in lactate with concomitant decrease in glucose concentrations. This combination can be observed in plasma of children with GA-I during acute encephalopathic crises. Underlying mechanisms may be the inhibition of the TCA cycle and/or respiratory chain with shift to lactate at the end of glycolysis, which is also supported by the 2-fold increase of the lactate/pyruvate ratio observed under 3-OHGA exposure. Lamp et al. have shown that 3-OHGA and GA inhibit astrocytic efflux and neuronal uptake of TCA cycle intermediates. These results suggest that elevated levels of 3-OHGA and GA may lead to neuronal injury and cell death via disruption of TCA cycle activity [21]. Direct effects on the respiratory chain have been reported controversially: While a recent report failed to prove changes on the activity of the different respi.Monium are produced by brain cells under the action of GA and 3-OHGA, suggesting a central liberation of ammonium in GA-I. Following the guidelines for GA-I, ammonium is not routinely determined during an acute illness [10,11], but could be worth to be measured in CSF. Ammonium is known to be toxic for brain cells causing reduced axonal elongation [16] as well as neuronal and oligodendrocytic cell death [15,18], which correlates with the brain atrophy and white matter changes observed in patients with primary hyperammonemias [20]. Its detection in brain cell cultures challenged with GA and 3-OHGA immediately raises the question of a potential role for ammonium in brain damage occurring in GA-I patients. As urea cycle is not active in central nervous system, ammonium produced during amino acid catabolism is mainly detoxified through amination of glutamate to glutamine by the enzyme glutamine synthetase. This enzyme is exclusivelyBrain Cell Damage in Glutaric Aciduria Type IFigure 5. Effects of GA and 3-OHGA on biochemical parameters measured in culture medium. Glucose (A), lactate (B), ammonium (C) and glutamine (D) were measured in the medium of cultures treated with protocols A (DIV 8) or B (DIV 14). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; *p,0.05, **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type IFigure 6. Evaluation of cell death after treatment with GA and 3-OHGA. (A; left panel) Immunohistochemical staining for cleaved caspase-3 (red signal). Scale bar: 100 mm. (A; right panel) Representative western blots with data quantification of whole-cell lysates for full length caspase-3 and the large fragment of cleaved (e.g. activated) caspase-3 for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of cleaved caspase-3 are expressed as percentage of respective controls. The values represent the mean 6 SEM from 3 replicates taken from 2 independent experiments. (B) In situ cell death assay with TUNEL (green signal) and cleaved caspase-3 (red signal) on DIV 8 (protocol A). Merge of both signals leads to double-stained cells appearing in yellow. Scale bar: 100 mm. (C) LDH in culture medium of cultures from protocol A (DIV 8, above) and protocol B (DIV 14, below). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type Isupported by the observation of neuronal loss in the Gcdh2/2 mouse model [13]. Analysis of media from treated and control cultures on DIV 14 showed a marked increase in lactate with concomitant decrease in glucose concentrations. This combination can be observed in plasma of children with GA-I during acute encephalopathic crises. Underlying mechanisms may be the inhibition of the TCA cycle and/or respiratory chain with shift to lactate at the end of glycolysis, which is also supported by the 2-fold increase of the lactate/pyruvate ratio observed under 3-OHGA exposure. Lamp et al. have shown that 3-OHGA and GA inhibit astrocytic efflux and neuronal uptake of TCA cycle intermediates. These results suggest that elevated levels of 3-OHGA and GA may lead to neuronal injury and cell death via disruption of TCA cycle activity [21]. Direct effects on the respiratory chain have been reported controversially: While a recent report failed to prove changes on the activity of the different respi.
