Neurotransmitter alterations in embryonic succinate semialdehyde dehydrogenase (SSADH) deficiency suggest a heightened excitatory state during development

Abstract

Background: SSADH (aldehyde dehydrogenase 5a1 (Aldh5a1); gamma-hydroxybutyric (GHB) aciduria) deficiency is a defect of GABA degradation in which the neuromodulators GABA and GHB accumulate. The human phenotype is that of nonprogressive encephalopathy with prominent bilateral discoloration of the globi pallidi and variable seizures, the latter displayed prominently in Aldh5a1-/- mice with lethal convulsions. Metabolic studies in murine neural tissue have revealed elevated GABA [and its derivatives succinate semialdehyde (SSA), homocarnosine (HC), 4,5-dihydroxyhexanoic acid (DHHA) and guanidinobutyrate (GB)] and GHB [and its analogue D-2-hydroxyglutarate (D-2-HG)] at birth. Because of early onset seizures and the neurostructural anomalies observed in patients, we examined metabolite features during Aldh5a1-/- embryo development.

Methods: Embryos were obtained from pregnant dams sacrificed at E (embryo day of life) 10-13, 14-15, 16-17, 18-19 and newborn mice. Intact embryos were extracted and metabolites quantified by isotope dilution mass spectrometry (n = 5-15 subjects, Aldh5a1+/+ and Aldh5a1-/-) for each gestational age group. Data was evaluated using the t test and one-way ANOVA with Tukey post hoc analysis. Significance was set at the 95th centile.

Results: GABA and DHHA were significantly elevated at all gestational ages in Aldh5a1-/- mice, while GB was increased only late in gestation; SSA was not elevated at any time point. GHB and D-2-HG increased in an approximately linear fashion with gestational age. Correlative studies in human amniotic fluid from SSADH-deficient pregnancies (n = 5) also revealed significantly increased GABA.

Conclusion: Our findings indicate early GABAergic alterations in Aldh5a1-/- mice, possibly exacerbated by other metabolites, which likely induce a heightened excitatory state that may predispose neural networks to epilepsy in these animals.

Figure 1

Schematic of GABA degradation and metabolites accumulating (arrows adjacent to metabolites) in Aldh5a1 deficiency (depicted by cross-hatched box). Abbreviations: SSA, succinate semialdehyde; α-KG, α-ketoglutarate; D-2-HG, D-2-hydroxyglutarate; DHHA, 4,5-dihydroxyhexanoic acid; TCA, tricarboxylic acid.

Figure 2

Concentrations of GABA, DHHA (4,5-dihydroxyhexanoic acid), SSA (succinate semialdehyde) and GB (guanidinobutyrate) in embryo extracts as a function of developmental age. Statistical analysis performed using a two-tailed t test. Abbreviations: gr, gram; E, embryonic (for example, 10–13 is equivalent to E10–E13); P, postnatal. Note that E 10–13 embryo extracts were not available for measurement of DHHA.

Figure 3

Concentrations of GHB (gamma-hydroxybutyric acid) and D-2-HG (D-2-hydroxyglutarate) in embryo extracts as a function of age. A linear representation of means ± SEM by genotype is depicted as well. Abbreviations and statistical analysis as described in Fig. 2. SEM, standard error of the mean.

Figure 4

Concentrations of glutamate and glutamine in embryo extracts as a function of age. Embryos were grouped as E15–17, 18–19 and day of life 1 (P1). Abbreviations: wt, wild-type (Aldh5a1^+/+ mice); mu, mutant (Aldh5a1^-/- mice). Statistical analysis performed using a two-tailed t test. One-way ANOVA with Tukey post-hoc analysis revealed a significant decline in glutamate level at birth as compared to E15–17 and E18–19, whereas glutamine levels were not significantly decreased.

Figure 5

GABA concentration in amniotic fluid derived from pregnancies carrying an affected SSADH-deficient human fetus. Data are depicted as box and whiskers, showing the range of values and quartiles. The box extends from the 25th centile to the 75th centile, with a line at the median (50th centile). Whiskers extend above and below the box to show the highest and lowest values. Statistical analysis performed using a two-tailed t test, n = 5 fluid samples for each group. Concentrations of other metabolites (DHHA, D-2-HG and SSA) were not significantly different as a function of pregnancy status.