Phenotype microarrays reveal metabolic dysregulations of neurospheres derived from embryonic Ts1Cje mouse model of Down syndrome

Abstract

Down syndrome (DS), is the most common cause of intellectual disability, and is characterized by defective neurogenesis during perinatal development. To identify metabolic aberrations in early neurogenesis, we profiled neurospheres derived from the embryonic brain of Ts1Cje, a mouse model of Down syndrome. High-throughput phenotypic microarray revealed a significant decrease in utilisation of 17 out of 367 substrates and significantly higher utilisation of 6 substrates in the Ts1Cje neurospheres compared to controls. Specifically, Ts1Cje neurospheres were less efficient in the utilisation of glucose-6-phosphate suggesting a dysregulation in the energy-producing pathway. T Cje neurospheres were significantly smaller in diameter than the controls. Subsequent preliminary study on supplementation with 6-phosphogluconic acid, an intermediate of glucose-6-phosphate metabolism, was able to rescue the Ts1Cje neurosphere size. This study confirmed the perturbed pentose phosphate pathway, contributing to defects observed in Ts1Cje neurospheres. We show for the first time that this comprehensive energetic assay platform facilitates the metabolic characterisation of Ts1Cje cells and confirmed their distinguishable metabolic profiles compared to the controls.

Fig 1. Distinctive patterns of substrate utilization between Ts1Cje and WT neurospheres.

Using plates PM-M1 through PM-M4, distinctive patterns of 48-hour substrate utilisation were observed between Ts1Cje and WT neurospheres, as indicated in red- and green-coloured boxes. The yellow boxes (well positions A1 to A3, in PM-M1 to PM-M4) represent the negative control wells, the black box (well positions H12, in PM-M2 to PM-M4) represents positive control well.

Fig 2. Normalised metabolic profiles of WT and Ts1Cje-derived neurospheres for plate PM-M1 to PM-M4.

(A) Normalised metabolic profiles of WT and Ts1Cje-derived neurospheres for plate PM-M1 to PM-M4. Each curve represents the metabolic signal produced in each well. The purple lines correspond to active profiles, whereas the yellow lines represent non-active profiles. The thick purple line represents the mean asymptote of active base curve. The x-axis and y-axis represent time in hours and normalised metabolic signal, respectively. (B) The pattern of the significantly utilised substrates changes with time captured at 6-hour intervals over the 48-hour incubation period.

Fig 3. Effect of 6PGA supplementation on neurosphere diameter.

(A) A healthy neurosphere is spherical in shape, with microspikes (red arrows) observed on the outer surface: magnification, X200. Neurospheres derived from cerebral cortices of WT (B) and Ts1Cje embryos (C), cultured at day 5 in vitro. Magnification, X100. The size of neurospheres derived from three biological replicates supplemented with 6PGA is represented in a scatter plot (D) (n = 288 vs 296, 227 vs 240 and 224 vs 249 for WT vs Ts1Cje for 0 mM, 1.0 mM and 2.0 mM 6PGA supplementations, respectively) whereas the mean measurement for each biological replicates is represented in a bar chart (E) (n = 3 per group per treatment). Each dot represents a neurosphere (D) or a biological replicate (E). Two-way ANOVA with Sidak’s multiple comparisons correction was performed on both analyses in (D) and (E). Black bars represent the mean diameter for each group. * denotes adjusted p<0.05 whereas *** denotes adjusted p<0.001.