Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I

Abstract

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

Figure 1. Treatment protocols.

Cultures of aggregates were exposed to 1 mM GA and 3-OHGA at two time points representing different developmental stages of brain cell maturation (Protocols A and B). Metabolites were added 6 times every 12 hours (indicated by arrows) starting on DIV 5 in protocol A and on DIV 11 in protocol B (treatment days are indicated by black boxes) 12 hours after the change of the medium. Aggregates were harvested 5 hours after the last treatment at DIV 8 in protocol A and at DIV 14 in protocol B.

Figure 2. Effects of GA and 3-OHGA on neurons.

(Left panel) Immunohistochemical staining for phosphorylated medium weight neurofilament (p-NFM) on cryosections of cultures derived from protocol A (DIV 8) and protocol B (DIV 14). Scale bar: 100 µm. (Right panel) Representative western blots with data quantification of whole-cell lysates for p-NFM for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of p-NFM are expressed as percentage of respective controls. The values represent the mean ± SD from 3 replicates taken from 2 independent experiments.

Figure 3. Effects of GA and 3-OHGA on astrocytes.

(Left panel) Immunohistochemical staining for glial fibrillary acidic protein (GFAP) on cryosections of cultures derived from protocol A (DIV 8) and protocol B (DIV 14). Swollen proximal fibers are indicated by black arrows. Scale bar: 100 µm. (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 ± SD from 3 replicates taken from 2 independent experiments.

Figure 4. Effects of GA and 3-OHGA on oligodendrocytes.

(Left panel) Immunohistochemical staining for galactocerebroside (GalC, DIV 8) and myelin basic protein (MBP, DIV 14) on cryosections derived from protocol A (DIV 8) and protocol B (DIV 14). Oligodendrocytes are indicated by black arrows. Scale bar: 100 µm. (Right panel) Representative western blots with data quantification of whole-cell lysates for MBP for protocol B (DIV 14). Actin was used as a loading control. The quantifications of MBP levels are expressed as percentage of respective controls. The values represent the mean ± SD from 3 replicates taken from 2 independent experiments.

Figure 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 ± SD of 7 replicate cultures assessed by Student’s t-test; *p<0.05, **p<0.01, *** p<0.001.

Figure 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 µm. (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 ± 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 µm. (C) LDH in culture medium of cultures from protocol A (DIV 8, above) and protocol B (DIV 14, below). Mean ± SD of 7 replicate cultures assessed by Student’s t-test; **p<0.01, *** p<0.001.

Figure 7. Expression of GCDH in neurons, astrocytes and oligodendrocytes.

In situ hybridization for GCDH mRNA in adult rat brain (16 µm cryosections), co-labeled by immunohistochemistry for specific markers of neurons (NeuN), astrocytes (GFAP) or oligodendrocytes (MBP). Top and central panels show expression of GCDH mRNA (blue signal) in cortical neurons (top; NeuN, red signal), while GCDH mRNA could not be detected in cortical astrocytes (central; GFAP, red signal, arrows pointing at astrocytic cell bodies). Bottom panel shows GCDH mRNA (blue signal) in granular neurons of cerebellum, while GCDH mRNA appears absent from adjacent oligodendrocytes in white matter of cerebellum (bottom; MBP, red signal). Scale bar: 100 µm.