Ltures derived from protocol A (DIV 8) and protocol B (DIV 14). Swollen proximal 61177-45-5 web fibers are indicated by black arrows. Scale bar: 100 mm. (Right panel) Representative western blots with data quantification of whole-cell lysates for GFAP for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of GFAP levels are expressed as percentage of respective controls. The values represent the mean 6 SD from 3 replicates taken from 2 independent experiments. 1676428 doi:10.1371/Tartrazine price journal.pone.0053735.gnumber of positively stained cells (e.g. apoptotic) in cultures exposed to GA and 3-OHGA in both treatment protocols as compared to control (Figure 6A, left panel). Accordingly, no significant changes were observed on activated caspase-3 level assessed by western blotting under both metabolites treatment at DIV 14. In protocol A, a tendency of activated caspase-3 to decrease in GA- and 3-OHGA-exposed aggregates was observed, however, the changes were not significant (Figure 6A, right panel). Interestingly, TUNEL labeling (green staining) showed an important signal increase for cultures treated with 3-OHGA on DIV 8, which only partially co-localized (yellow staining) with cleaved caspase-3-labeled apoptotic cells (red signal) (Figure 6B). This suggests an induction of non-apoptotic cell death in developing brain cells under 25837696 3-OHGA exposure. TUNEL signal in 3-OHGA exposed cultures was homogenously distributed over the entire aggregate, suggesting that 3-OHGA diffused well from the medium into the whole aggregate (Figure 6B, 106magnifications).DiscussionWe used 3D organotypic brain cell cultures in aggregates to explore, in vitro, the effects of the two main metabolites (GA and 3OHGA) accumulated in body fluids of subjects affected by GA-I. Our in vitro model is particularly suitable for studying neurotoxicity because the aggregates contain all types of brain cells with their spontaneous connections between each other. In addition, this model reproduces early phases of brain development and has proven to be optimal to study differential effects of metabolic derangements (such as hyperammonemia) in developing brain compared to adult brain tissue [14,15,16,18]. This is particularly important when studying a disorder in which the most dramatic brain damage occurs in early childhood. In our study, we could confirm that both GA and 3-OHGA were deleterious for brain cells during development, but 3-OHGA turned out to be the most toxic metabolite. The most striking effect of 3-OHGA on the aggregates was massive tissue destruction with wide areas of cell death, which was evident on DIV 14 (Figures 2 and 3). In both developmental stages (DIV 8 and 14) astrocytes appeared more susceptible to GA and 3-OHGA toxicity than neurons and oligodendrocytes, showing important morphological changes (decrease in fiber density and swelling of proximal fibers). These results are in line with the findings in the Gcdh2/2 mouse which under high lysine diet show reactive astrocytes [13]. Surprisingly, neurons did not show any significant alteration in our model. This might be due to their origin from wild type animals,GCDH Expression in Adult Rat BrainGcdh mRNA was found expressed in the whole rat brain, exclusively in neurons (Figure 7). No Gcdh mRNA was detectable in astrocytes or in oligodendrocytes.Brain Cell Damage in Glutaric Aciduria Type IFigure 4. Effects of GA and 3-OHGA on oligodendrocytes. (Left panel) Immunohistochemi.Ltures derived from protocol A (DIV 8) and protocol B (DIV 14). Swollen proximal fibers are indicated by black arrows. Scale bar: 100 mm. (Right panel) Representative western blots with data quantification of whole-cell lysates for GFAP for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of GFAP levels are expressed as percentage of respective controls. The values represent the mean 6 SD from 3 replicates taken from 2 independent experiments. 1676428 doi:10.1371/journal.pone.0053735.gnumber of positively stained cells (e.g. apoptotic) in cultures exposed to GA and 3-OHGA in both treatment protocols as compared to control (Figure 6A, left panel). Accordingly, no significant changes were observed on activated caspase-3 level assessed by western blotting under both metabolites treatment at DIV 14. In protocol A, a tendency of activated caspase-3 to decrease in GA- and 3-OHGA-exposed aggregates was observed, however, the changes were not significant (Figure 6A, right panel). Interestingly, TUNEL labeling (green staining) showed an important signal increase for cultures treated with 3-OHGA on DIV 8, which only partially co-localized (yellow staining) with cleaved caspase-3-labeled apoptotic cells (red signal) (Figure 6B). This suggests an induction of non-apoptotic cell death in developing brain cells under 25837696 3-OHGA exposure. TUNEL signal in 3-OHGA exposed cultures was homogenously distributed over the entire aggregate, suggesting that 3-OHGA diffused well from the medium into the whole aggregate (Figure 6B, 106magnifications).DiscussionWe used 3D organotypic brain cell cultures in aggregates to explore, in vitro, the effects of the two main metabolites (GA and 3OHGA) accumulated in body fluids of subjects affected by GA-I. Our in vitro model is particularly suitable for studying neurotoxicity because the aggregates contain all types of brain cells with their spontaneous connections between each other. In addition, this model reproduces early phases of brain development and has proven to be optimal to study differential effects of metabolic derangements (such as hyperammonemia) in developing brain compared to adult brain tissue [14,15,16,18]. This is particularly important when studying a disorder in which the most dramatic brain damage occurs in early childhood. In our study, we could confirm that both GA and 3-OHGA were deleterious for brain cells during development, but 3-OHGA turned out to be the most toxic metabolite. The most striking effect of 3-OHGA on the aggregates was massive tissue destruction with wide areas of cell death, which was evident on DIV 14 (Figures 2 and 3). In both developmental stages (DIV 8 and 14) astrocytes appeared more susceptible to GA and 3-OHGA toxicity than neurons and oligodendrocytes, showing important morphological changes (decrease in fiber density and swelling of proximal fibers). These results are in line with the findings in the Gcdh2/2 mouse which under high lysine diet show reactive astrocytes [13]. Surprisingly, neurons did not show any significant alteration in our model. This might be due to their origin from wild type animals,GCDH Expression in Adult Rat BrainGcdh mRNA was found expressed in the whole rat brain, exclusively in neurons (Figure 7). No Gcdh mRNA was detectable in astrocytes or in oligodendrocytes.Brain Cell Damage in Glutaric Aciduria Type IFigure 4. Effects of GA and 3-OHGA on oligodendrocytes. (Left panel) Immunohistochemi.
be optimal to study differential effects of metabolic derangements (such as hyperammonemia) in developing brain compared to adult brain tissue [14,15,16,18]. This