Fig. 4

Increased glutamate uptake and metabolism in FTD3 astrocytes. a Representative immunocytochemistry images of astrocytes from FTD3 patient cell lines (H150 and H151) and their respective isogenic control cell lines at 10 weeks of maturation. All cell lines express astrocyte markers Aquaporin-4 (AQP4), S100beta (S100β) and SOX9. Scale bar: 25 μm. b Percentage distribution of ¹³C-labelled metabolites obtained from direct metabolism [U-¹³C] glutamate in FTD3 astrocytes from the patient cell lines (white bars) and their respective isogenic controls (green bars). Astrocytes in culture were incubated for 90 min with [U-¹³C] glutamate (0.25 mM) in the presence of unlabelled glucose (2.5 mM): astrocyte extracts were subsequently collected and analysed using GC-MS for determination of ¹³C-enrichment. The increased labelling in the intracellular glutamate and other TCA cycle metabolites and amino acids after [U-¹³C] glutamate incubation indicates increased uptake and metabolism of this amino acid. Results represent means ± S.E.M. obtained from three different patient cell lines from three different culture preparations of hiPSC-derived neurons. *P < 0.05 or **P < 0.001; two-way ANOVA correcting for multiple comparisons was employed. c Representative Western blot analyses of protein expression of glutamine synthetase (GS). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Expression of GS protein was found to be upregulated in FTD3 neurons from the two different patient cell lines (H150 and H151)