The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

Ammonium accumulation and chemokine decrease in culture media of Gcdh-/- 3D reaggregated brain cell cultures.

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain. We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh-/- mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys). Significant amounts of GA and 3-OHGA were detected in Gcdh-/- aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh-/- aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh-/- aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh-/- aggregates at DIV 14 and after exposure to Lys at DIV 8. This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh-/- brain cells. We described for the first time a decrease of chemokines in Gcdh-/- culture media which might contribute to brain cell injury in GA-I.

New in vitro model derived from brain-specific Mut-/- mice confirms cerebral ammonium accumulation in methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria (MMAuria) is an inborn error of metabolism leading to neurological deterioration. In this study, we used 3D organotypic brain cell cultures derived from embryos of a brain-specific Mut-/- (brain KO) mouse to investigate mechanisms leading to brain damage. We challenged our in vitro model by a catabolic stress (temperature shift). RESULTS: Typical metabolites for MMAuria as well as a massive NH4+ increase were found in the media of brain KO cultures. We investigated different pathways of intracerebral NH4+ production and found increased expression of glutaminase 2 and diminished expression of GDH1 in Mut-/- aggregates. While all brain cell types appeared affected in their morphological development in Mut-/- aggregates, the most pronounced effects were observed on astrocytes showing swollen fibers and cell bodies. Inhibited axonal elongation and delayed myelination of oligodendrocytes were also noted. Most effects were even more pronounced after 48 h at 39 °C. Microglia activation and an increased apoptosis rate suggested degeneration of Mut-/- brain cells. NH4+ accumulation might be the trigger for all observed alterations. We also found a generalized increase of chemokine concentrations in Mut-/- culture media at an early developmental stage followed by a decrease at a later stage. CONCLUSION: We proved for the first time that Mut-/- brain cells are indeed able to produce the characteristic metabolites of MMAuria. We confirmed significant NH4+ accumulation in culture media of Mut-/- aggregates, suggesting that intracellular NH4+ concentrations might even be higher, gave first clues on the mechanisms leading to NH4+ accumulation in Mut-/- brain cells, and showed the involvement of neuroinflammatory processes in the neuropathophysiology of MMAuria.

Immunolocalization of glutaryl-CoA dehydrogenase (GCDH) in adult and embryonic rat brain and peripheral tissues.

Glutaryl-CoA dehydrogenase (GCDH) is a mitochondrial enzyme that is involved in the degradation of tryptophan, lysine and hydroxylysine. Deficient enzyme activity leads to glutaric aciduria type-I (GA-I). This neurometabolic disease usually manifests with acute encephalopathic crises and striatal neuronal death in early childhood leading to an irreversible dystonic-dyskinetic movement disorder. Fronto-temporal atrophy and white matter changes are already present in the pre-symptomatic period. No detailed information on GCDH expression during embryonic development and in adulthood was available so far. Using immunofluorescence microscopy and cell-type-specific markers to localize GCDH in different tissues, we describe the differential cellular localization of GCDH in adult rat brain and peripheral organs as well as its spatiotemporal expression pattern. During embryonic development GCDH was predominantly expressed in neurons of the central and peripheral nervous system. Significant expression levels were found in epithelial cells (skin, intestinal and nasal mucosa) of rat embryos at different developmental stages. Besides the expected strong expression in liver, GCDH was found to be significantly expressed in neurons of different brain regions, renal proximal tubules, intestinal mucosa and peripheral nerves of adult rats. GCDH was found widely expressed in embryonic and adult rat tissues. In rat embryos GCDH is predominantly expressed in brain implying an important role for brain development. Interestingly, GCDH was found to be significantly expressed in different other organs (e.g. kidney, gut) in adult rats probably explaining the evolving phenotype in GA-I patients.

Ammonium accumulation is a primary effect of 2-methylcitrate exposure in an in vitro model for brain damage in methylmalonic aciduria.

Using 3D organotypic rat brain cell cultures in aggregates we recently identified 2-methylcitrate (2-MCA) as the main toxic metabolite for developing brain cells in methylmalonic aciduria. Exposure to 2-MCA triggered morphological changes and apoptosis of brain cells. This was accompanied by increased ammonium and decreased glutamine levels. However, the sequence and causal relationship between these phenomena remained unclear. To understand the sequence and time course of pathogenic events, we exposed 3D rat brain cell aggregates to different concentrations of 2-MCA (0.1, 0.33 and 1.0mM) from day in vitro (DIV) 11 to 14. Aggregates were harvested at different time points from DIV 12 to 19. We compared the effects of a single dose of 1mM 2-MCA administered on DIV 11 to the effects of repeated doses of 1mM 2-MCA. Pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh were used to block apoptosis. Ammonium accumulation in the culture medium started within few hours after the first 2-MCA exposure. Morphological changes of the developing brain cells were already visible after 17h. The highest rate of cleaved caspase-3 was observed after 72h. A dose-response relationship was observed for all effects. Surprisingly, a single dose of 1mM 2-MCA was sufficient to induce all of the biochemical and morphological changes in this model. 2-MCA-induced ammonium accumulation and morphological changes were not prevented by concomitant treatment of the cultures with pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh: ammonium increased rapidly after a single 1mM 2-MCA administration even after apoptosis blockade. We conclude that following exposure to 2-MCA, ammonium production in brain cell cultures is an early phenomenon, preceding cell degeneration and apoptosis, and may actually be the cause of the other changes observed. The fact that a single dose of 1mM 2-MCA is sufficient to induce deleterious effects over several days highlights the potential damaging effects of even short-lasting metabolic decompensations in children affected by methylmalonic aciduria.