Fig 1.
Kaplan-Meier plots of ammonium acetate-induced mortality.
Zebrafish larvae (n = 100 per group) at 1 or 4 dpf were exposed to varying concentrations (5, 10 and 20 mM) of either NH4Ac or NaAc and survival rates monitored for up to 36 h. While NH4Ac was not toxic to zebrafishes at 1 dpf (A), exposure at 4 dpf induced death of all larvae in a dose-dependent manner (log-rank test, P<0.001) (B). In contrast, NaAc had virtually no toxic or lethal effect at either developmental stage (C, D).
Fig 2.
Cerebral expression patterns of glutamine synthetase isoforms during zebrafish development.
Localization of mRNAs encoding for the glutamine synthetases GLULA, GLULB and GLULC in the CNS of zebrafish larvae as visualized by ISH. Pictures are representative images of 3 independent experiments (n = 50 embryos/larvae per stage and experiment). Images show the embryonic heads at stages indicated. Left columns of each row show latter with anterior to the left and right columns with anterior to the top; FB, forebrain; MB, midbrain. Glula was expressed in radial glia (arrowheads) starting at 3 dpf with constantly increasing expression up to 5 dpf (A). Glulb was expressed in neural crest and neuromast cells (arrowheads) (B), Glulc expression was restricted to distinct cells of the nostrils (arrowheads) throughout indicated stages (C).
Fig 3.
Acute hyperammonemia induces expression of specific glutamine synthetase and glutaminase isoforms.
Zebrafish larvae at 1 or 4 dpf were exposed to 10 mM of either NH4Ac or NaAc and expression of indicated mRNAs analyzed using qRT-PCR. While NH4+-exposure did not change expression of glutamine synthetase (Glula, Glulb and Glulc) or glutaminase (Glsa, Gls2b) isoforms at 1 dpf, exposure at 4 dpf induced gene expression of Glula, Glulb, Glsa and Gls2b, respectively (A, B). Data are expressed as mean +/- SD in fold-change (whole body lysates, n = 3 with 50 larvae per group and experiment; ANOVA, *P<0.05).
Fig 4.
Exposure to NH4Ac leads to strong increase in glutamine and glutamate concentrations and depletion of ornithine and alanine.
Zebrafish larvae at 1 or 4 dpf were exposed to 10 mM NH4Ac for 6 or 12 h and amino acids content in whole body lysates determined by HPLC. While larvae at 1 dpf showed increased urea production after 12 h of NH4-exposure when compared to the non-exposed control cohort (B), exposed zebrafishes at 4 dpf exhibited significantly increased glutamine and glutamate concentrations after 6 and 12 h, respectively (C, D). Simultaneously, NH4-exposure caused a significant depletion of branched-chain amino acids, alanine, ornithine and subsequently arginine at 4 dpf (D). Data are expressed as mean +/- SD in μmol per g protein (whole body lysates, n = 3 with 50 larvae per group and experiment; Student’s t-test, *P<0.05, **P<0.01).
Fig 5.
Transamination via ornithine and alanine aminotransferases are pivotal processes in acute hyperammonemia.
Zebrafish larvae at 4 dpf were exposed to 10 mM NH4Ac or NaAc for 6 or 12 h and expression of indicated mRNAs analyzed using qRT-PCR. Whole body lysates of exposed zebrafish embryos were subjected to quantitative analysis of pyruvate, lactate or 2-oxoglutarate concentrations as described under Materials and Methods. NH4-exposure induced gene expression of alanine aminotransferase isoform Gpt2 and Oat, while Gpt expression was unchanged (A-C). Exposure to NaAc had no effect on the expression of either aminotransferase (A-C). Larvae exposed to NH4Ac displayed increased pyruvate and lactate concentrations when compared to the control cohort (D, E). In addition, 2-oxoglutarate concentrations were significantly depleted upon NH4-exposure after 6 h, with a further decline after 12 h (F). Data are expressed as mean +/- SD in fold-change (A-C) or μmol per g protein (D-F) (whole body lysates, n = 3 with 50 larvae per group and experiment; ANOVA, *P<0.05, **P<0.01).
Fig 6.
Glutamatergic signaling via NDMA receptors mediates neurotoxicity in acute hyperammonemia.
Endogenous mRNA expression of zebrafish homologs of NDMA receptor subunits (Grin1a, Grin1b, Grin2ab, Grin2da and Grin3a) during zebrafish development (50% epiboly to 5 dpf). Expression of Grin1a, Grin1b, Grin2da and Grin3a strongly increased at 5 dpf (A), coinciding with the time point of NH4-induced mortality. Larvae exposed to 10 mM NH4Ac at 4 dpf displayed a downregulated gene expression of NMDA receptor subunits Grin1a, Grin1b and Grin2da after 6 and 12 h, respectively (B). Data are expressed as mean +/- SD in fold-change (whole body lysates, n = 3 with 50 larvae per group and experiment; ANOVA, *P<0.05, **P<0.01).
Fig 7.
Comparison of different therapeutic concepts: OAT-inhibition prevents mortality in acute hyperammonemia and rescues brain cell death.
Zebrafish larvae (n = 100 per group) at 4 dpf were treated with L-MSO (25 μM), ketamine (50 μM), or memantine (50 μM) alone or in combination or with increasing concentrations of 5-FMO (50 to 200 μM) for 30 minutes following exposure to 10 mM NH4Ac. While mock-treated zebrafish larvae died within 21 h (median survival: 17 h), larvae preincubated with L-MSO or ketamine succumbed to 10 mM NH4Ac within 24 h (median survival: 21.5 h; log-rank test, P<0.01) or 25 h (median survival: 22 h; log-rank test, P<0.01), respectively. Treatment with memantine prolonged survival up to 27 h (median survival 22h; log-rank test, P<0.01) (A). Combination of L-MSO and memantine did not further increase survival rates as compared to the treatment with memantine alone (B). Treatment with 50 μM 5-FMO for 30 min prior to NH4-exposure prolonged survival up to 31 h (median survival: 27 h; log-rank test, P<0.0001). Further dosage escalation had an incremental benefit on survival rates. While 72% of larvae treated with 100 μM 5-FMO survived the observation period of 36 h of NH4Ac exposure, treatment with 200 μM 5-FMO increased the survival rate of exposed larvae to 91% (C). Representative illustration of zebrafishes at 4 dpf either treated with 200 μM 5-FMO or mock (w/o inhibitor) for 30 min prior to exposure to 10 mM NH4Ac for 12 h (n = 3 with 50 larvae per group and experiment) (D). Zebrafishes were fixed with 5% PFA in PBS overnight and imaged using a binocular microscope (MZ16 F, Leica). Pictures show larvae with anterior to the right. Exposure to 10 mM NH4Ac induced brain cell death (asterix), which was rescued by treatment with 5-FMO in a concentration of 200 μM as compared to the non-exposed control cohort (D). Quantitative analysis of larvae exhibiting signs of brain damage in the different groups (E). Data are expressed as mean +/- SD in % of total exhibiting microscopic signs of brain cell death (n = 3 with 50 larvae per group and experiment; ANOVA, **P<0.01).
Fig 8.
OAT-inhibition corrects biochemical alterations and NMDA receptor expression in acute hyperammonemia.
Zebrafish larvae at 4 dpf were treated with increasing concentrations of 5-FMO (50 μM to 200 μm) for 30 min followed by exposure to 10 mM NH4Ac for 12 h. Thereafter, zebrafishes were lysed and either subjected to HPLC for determination of amino acids (A, B) or RNA extraction and subsequent qRT-PCR analysis (C-E). Treatment with 5-FMO led to a normalization of glutamate and glutamine concentrations (A) and restored ornithine and arginine concentrations (B) in a dose-dependent manner. Moreover, mRNA expression levels of NMDA receptor subunits Grin1a and Grin1b normalized upon treatment with 200 μm 5-FMO (C, D). Data are expressed as mean +/- SD in μmol per g protein (A, B) or in fold-change (C-E) (whole body lysates, n = 3 with 50 larvae per group and experiment; ANOVA, *P<0.05, **P<0.01).