Fig 1.
Interrelationships of GABA metabolism, and the effect of GABA on mTOR (mechanistic target of rapamycin).
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.
Fig 2.
Nissl staining of neural stem cells (NSCs) and cell culture medium concentration of GHB as a function of cell line genotype.
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 [29]. 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).
Fig 3.
Fluorescence detection of cellular toxicity as a function of starvation or treatment with the mTOR inhibitor, XL-765.
NSCs were exposed to vehicle (0.01% DMSO) or XL-765 (see Methods) for 24 h, then starvation conditions were implemented for 6 h followed by fluorescence quantification to assess cytotoxicity. Each symbol (square) represents the measurement of each biological replicate. Each horizontal line reflects the mean of the 3 independent experiments and error bars represent SD. When present, XL-765 was used at 10 nM in DMSO (0.01%) for 24 h, otherwise vehicle matched controls were employed. Note that a lower value within genotype represents decreased cytotoxicity. Abbreviations: RFU, relative fluorescence units; WT, wild-type; MT, mutant; VEH (0.01% DMSO); XL-765, treatment with XL-765. Statistical analysis employed one-way ANOVA with post-hoc analysis (F (3, 8) = 4.895; p < 0.05).
Fig 4.
Mitochondrial abundance and oxidative stress parameters in NSCs.
MitotrackerTM 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)). CellROXTM 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 (MitoSoxTM 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 Fig 3.
Fig 5.
Relative ATP consumption rates and initial ATP concentration in NSCs.
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 Fig 4A were analyzed using two-way ANOVA for half-life, as well as effects of time and treatment/genotype. This revealed interaction (half-lives;t1/2 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 3 and 4.
Fig 6.
Selected genes dysregulated in NSCs as compared to homogenates of isolated hypothalami.
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) aldh5a1+/+ (white boxes) and aldh5a1-/- (black boxes) mice. Shown are Gabrb3, the β3 subunit of the GABAB receptor, solute carriers (Slc) 1a2, 12a2 and 12a5, 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 GABAB receptor was consistent regardless of tissue or age. Statistical analysis, Student’s two-tailed t test; *p<0.05; **p<0.01; ***p<0.001.
Table 1.
Reagents employed for in vitro characterization of neural stem cells.