Characterization of [3H]-D-Aspartate uptake in astrocytes differentiated from human induced pluripotent stem cells of patients with amyotrophic lateral sclerosis

Astrocytes are responsible for a large part of glutamate uptake; in amyotrophic lateral sclerosis (ALS) and other diseases, dysfunction of this system is observed. The study of glutamate uptake in patients with ALS is essential to understand the excitotoxicity of glutamate in this disease. We aim to characterize the [3H]-D-Aspartate uptake profile of differentiated astrocytes from induced pluripotent stem cells (IPSC) derived from Brazilian patients with ALS and healthy individuals. [3H]-D-Aspartate uptake assay was conducted to analyze cell functionally, evaluating baseline levels and the effect of the glutamate transporter blockers, DL-TBOA, and DHK (10, 25, 50, and 100 µM). A control cell line (CTRL) and an ALS cell line were used in analyzes at 3 and 6 weeks of in vitro culture (WIV). Statistical analysis was performed using the GraphPad prism 8.4.3 software. We observed a reduction of 33% in 3WIV and 41% in 6WIV of uptake in the ALS line in relation to control. In 3WIV, blockade with DL-TBOA significantly reduced uptake in the CTRL line at all concentrations used, while no effect was observed in the ALS line. In 6WIV, blockade with DL-TBOA significantly reduced uptake in the CTRL lineage at all concentrations used, while in the ALS lineage the blockage could be observed at concentrations of 25 and 100 µM. In preliminary experiments with DHK, a selective blocker of GLT1, we observed, in 3WIV, a tendency towards reduced uptake at concentrations of 25, 50 and 100 µM in CTRL astrocytes, while in the ALS strain it was only observed at concentrations of 25 and 100 µM.  In 6WIV we observed a tendency to block uptake by DHK at concentrations of 25, 50 and 100 µM in CTRL astrocytes. Our results demonstrate that differentiated astrocytes from IPSC have a functional [3H]-D-Aspartate uptake system. Furthermore, we can suggest that there is an uptake dysfunction in cells of the ALS lineage. FAPERJ, CAPES, CNPq