Relative ATP consumption rates and initial ATP concentration in NSCs.

<p>ATP concentrations were detected with an ATP-sensitive luminescent probe and monitored for 65 minutes to evaluate the rate of ATP consumption (A; indicating relative concentrations at that time point). Initial relative ATP concentration (B) between genotype and treatment. Data represents the mean of 3 replicates and error bars represent SD. XL-765 was employed at 10 nM in vehicle (Veh; 0.01% DMSO) for 24 h. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186919#pone.0186919.g004" target="_blank">Fig 4A</a> were analyzed using two-way ANOVA for half-life, as well as effects of time and treatment/genotype. This revealed interaction (half-lives;t_1/2 in min.) for ATP consumption that were not significantly different between mutant (starvation conditions, 58.6 [50.6–69.5] and XL-765, 56.0 [44.4–75.7]) and wild-type (starvation conditions, 56.0 [48.9–65.2] and XL-765, 58.6 [48.1–75.0]) following 24 hr of growth/treatment (F (36, 104) = 0.8699; p = ns). Conversely, effects of time and treatment/genotype were significant (time, F (12, 104) = 41.85, p<0.0001; treatment/genotype, F (3, 104) = 177.4; p<0.0001). Data for Fig 5B was evaluated with a one-way ANOVA and post hoc analysis (F (3, 12) = 56.19; p<0.0001. Abbreviations employed were as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186919#pone.0186919.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186919#pone.0186919.g004" target="_blank">4</a>.</p

Nissl staining of neural stem cells (NSCs) and cell culture medium concentration of GHB as a function of cell line genotype.

<p>To confirm that NSCs retained features of neural cells, Nissl stain (red) was initially undertaken, and co-localization with a nuclear stain (blue) was performed to confirm that cells were of neuronal origin (A), although the Nissl stain does not differentiate mature neurons from progenitors. Scale bar = 50 μm (white bar). GHB concentration in cell culture medium is also displayed as a function of donor cell line (B). Media were collected for 24 h following addition of fresh medium and quantified for GHB content employing isotope-dilution gas chromatography-mass spectrometry [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186919#pone.0186919.ref029" target="_blank">29</a>]. GHB content was normalized to the recovery of ²H₆-GHB, the latter added to each medium sample as internal standard stable isotope. The lower limit of quantification for this assay was 30 μM; all wild-type samples were below the limit of quantification (BLQ). Error bars display mean and SD. Abbreviations: WT, wild-type; MT, mutant. Statistical analysis employed one-way ANOVA (F (3, 42) = 378.9; p < 0.0001).</p

<i>In vitro</i> modeling of experimental succinic semialdehyde dehydrogenase deficiency (SSADHD) using brain-derived neural stem cells

<div><p>We explored the utility of neural stem cells (NSCs) as an <i>in vitro</i> model for evaluating preclinical therapeutics in succinic semialdehyde dehydrogenase-deficient (SSADHD) mice. NSCs were obtained from <i>aldh5a1</i>^<i>+/+</i> and <i>aldh5a1</i>^<i>-/-</i> mice (aldh5a1 = aldehyde dehydrogenase 5a1 = SSADH). Multiple parameters were evaluated including: (1) production of GHB (γ-hydroxybutyrate), the biochemical hallmark of SSADHD; (2) rescue from cell death with the dual mTOR (mechanistic target of rapamycin) inhibitor, XL-765, an agent previously shown to rescue <i>aldh5a1</i>^<i>-/-</i> mice from premature lethality; (3) mitochondrial number, total reactive oxygen species, and mitochondrial superoxide production, all previously documented as abnormal in <i>aldh5a1</i>^<i>-/-</i> mice; (4) total ATP levels and ATP consumption; and (5) selected gene expression profiles associated with epilepsy, a prominent feature in both experimental and human SSADHD. Patterns of dysfunction were observed in all of these parameters and mirrored earlier findings in <i>aldh5a1</i>^<i>-/-</i> mice. Patterns of dysregulated gene expression between hypothalamus and NSCs centered on ion channels, GABAergic receptors, and inflammation, suggesting novel pathomechanisms as well as a developmental ontogeny for gene expression potentially associated with the murine epileptic phenotype. The NSC model of SSADHD will be valuable in providing a first-tier screen for centrally-acting therapeutics and prioritizing therapeutic concepts of preclinical animal studies applicable to SSADHD.</p></div

Interrelationships of GABA metabolism, and the effect of GABA on mTOR (mechanistic target of rapamycin).

<p>Upward arrows indicate elevations of metabolites seen in both human and murine SSADHD. Bars indicate blockade. Abbreviations: GAD, glutamate decarboxylase; GABA-T, GABA-transaminase; SSA, succinic semialdehyde; GHB, γ-hydroxybutyrate; SSADH, succinic semialdehyde dehydrogenase; NADH, reduced nicotinamide dinucleotide.</p

Mitochondrial abundance and oxidative stress parameters in NSCs.

<p>Mitotracker^TM fluorescent staining was employed to assess mitochondrial abundance.Quantitation of fluorescent microscopy images in which MitoTracker signal (green) was normalized to nuclear stain (blue, NucBlue) (A) and representative fluorescence images employed for data accrual (B; scale bar = 50 μm (white)). CellROX^TM staining was used to assess overall degree of oxidative stress (total reactive oxygen species), and different durations of treatment with starvation media (12–36 hours) (C). Mitochondrial specific oxidative stress was determined with a luminescence assay (MitoSox^TM system), in the presence and absence of 10 nM XL-765 (normalized to cell viability) (D). All values represent the mean of 3 replicates with error bars denoting SD. Data for Fig 4A assessed using a two-tailed t test. For Fig 4C, a one-way ANOVA with post-hoc analysis was used within genotype (wild-type, (F (2, 51) = 12.5; p = ns: mutant, F (2, 51) = 12.5; p<0.0001) and for Fig 4D across genotypes (F (3, 8) = 10.72, p<0.01). Abbreviations employed were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186919#pone.0186919.g003" target="_blank">Fig 3</a>.</p

Selected genes dysregulated in NSCs as compared to homogenates of isolated hypothalami.

<p>Neural stems cells were harvested following 24 h of culture in 6-well plates; hippocampi were dissected from P1 and P20 (postnatal day of life) <i>aldh5a1</i>^<i>+/+</i> (white boxes) and <i>aldh5a1</i>^<i>-/-</i> (black boxes) mice. Shown are <i>Gabrb3</i>, the β3 subunit of the GABA_B receptor, solute carriers (<i>Slc</i>) <i>1a2</i>, <i>12a2</i> and <i>12a5</i>, respectively, representing family members of the solute carrier organic acid transporter family, and tumor necrosis factor α (TNFα). Gene expression was measured via quantitative reverse transcription-polymerase chain reaction (RT-PCR). NSC expression levels were normalized to wild-type NSCs, and hippocampal extract results were normalized to P20 wild-type mice. Bars denote the mean ± SD of 3–4 biological replicates. ND, denotes that expression was not detectable. Note the developmental differences in the solute carrier transporters as a function of both tissue age and NSCs, underscoring a high degree of ontogeny. Conversely, up-regulation of the β3 subunit of the GABA_B receptor was consistent regardless of tissue or age. Statistical analysis, Student’s two-tailed <i>t</i> test; *p<0.05; **p<0.01; ***p<0.001.</p

Reagents employed for in vitro characterization of neural stem cells.

<p>Reagents employed for in vitro characterization of neural stem cells.</p

Western blot.

<p>Lysates of knock-out homozygous <i>aldh7a1</i> zebrafish embryos (at 8 dpf) western blot analysis showing no aldh7a1 protein compared to wild-type.</p

EEG results of knock-out homozygous <i>aldh7a1</i> embryos and wild type embryos at 9 dpf.

<p>A) This shows spike discharges in a homozygous with no response to 100 μM diazepam, but almost normalization of EEG on 25 mM pyridoxine compared to wild type. B) This shows another homozygous with no response to phenobarbital, but almost normalization of EEG on pyridoxine (25 mM) compared to wild type. C) This shows normal EEG with spikes on pentylenetetrazole, and normalization of EEG on diazepam.</p

Daily survival of heterozygous 5 bp deletion male and female zebrafish embryos.

<p>Y axis to the left indicates total number of embryos in the tank. Y axis to the right indicates the number of dead embryos genotyped and presented as a histogram. X axis shows dpf.</p