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. 2022 Jun 6;4(3):fcac144. doi: 10.1093/braincomms/fcac144

Figure 4.

Figure 4

4-Phenylbutyrate acid (PBA) rescued the GABA uptake function in the patient astrocytes and neurons. (A) Human astrocytes and inhibitory neurons were differentiated from the neural progenitor cells (NPCs) from the patient and the CRISPR corrected isogenic control line. Live images of human NPCs, astrocytes and GABAergic inhibitory neurons differentiated from the human-induced pluripotent stem cells (iPSCs) on the day of experiment. (B–E) Astrocytes at Days 30–35 (B, C) or neurons at Days 60–65 (D, E) after differentiation were treated with PBA (2 mM) for 24 h before 3H radioactive GABA uptake assay. The DMSO-treated corrected or patient cells were taken as 1. (F) Corrected astrocytes at Days 30–35 after differentiation were transfected with the wildtype or the mutant GAT-1YFP cDNAs (1 µg per a 35 mm2 dish) for 48 h before 3H radioactive GABA uptake assay. PBA (2 mM) was applied for 24 h before GABA uptake assay experiment. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/µg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was DMSO-treated condition taken as 1. In B, D and F, 966 stands for Cl-966 (50 µM) and 711 stands for NNC-711 (35 µM) that was applied 30 min to the astrocytes transfected with the wildtype GAT-1YFP before preincubation and removed during preincubation. In B and D, ***P < 0.001 versus corrected DMSO treated. §§<0.01, §§§P < 0.001 versus patient DMSO treated. In C and E, the PBA-treated corrected or patient GABA uptake was normalized to its DMSO treated. In C, E and F, the PBA-treated corrected/wildtype or patient/mutant GABA uptake was normalized to its DMSO treated. *P < 0.05; **P < 0.01; ***P < 0.001 versus DMSO treated in its own group. In B, C, D and E, n = 4–8 batches of cells. In F, n = 4–5 different transfections. Unpaired t test was used for C and E. One-way analysis of variance followed by a Dunnett post hoc multiple comparison test was used in B, D and F. Values were expressed as mean ± SEM.