Astrocytic EAAT Dysfunction and Inflammatory Dysregulation in Amyotrophic Lateral Sclerosis

Introduction: Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive motor neuron loss associated with glutamatergic excitotoxicity and dysfunction of EAAT1/EAAT2 transporters. Neuroinflammation may further modulate astrocyte function through cytokines such as TNF-α, IL-1α, and IL-1β. Objective: To investigate EAAT1/EAAT2 dysfunction and astrocyte reactivity in iPSC-derived astrocytes from ALS patients. Methods: Three control (CTRL) and three ALS-derived astrocyte lines (familial SOD1, familial VAPB, and sporadic VAPB) were characterized by immunocytochemistry and western blot. [³H]-D-Aspartate uptake was assessed in the presence of DL-TBOA and DHK and following TNF-α, IL-1α, or IL-1β treatment. TNF-α levels were measured by ELISA, while IL-1α and IL-1β expression was assessed by RT-qPCR. Results: All cell lines were positive for GFAP and vimentin. Glutamate uptake was reduced in ALS astrocytes compared with CTRLs (F(5,151)=12.31; P<0.0001), with lower uptake in SOD1 than familial VAPB astrocytes (P=0.0190). DL-TBOA reduced uptake in CTRLs and ALS lines, although higher concentrations were required in SOD1 astrocytes. DHK reduced uptake in CTRLs, while its effect was markedly attenuated in ALS lines and absent in sporadic VAPB. SOD1 astrocytes showed reduced EAAT1 (F(2,7)=18.26; P=0.0017) and increased total EAAT2 density (t=3.37; P=0.0149). TNF-α reduced uptake across groups and was endogenously increased in ALS astrocytes (F(2,12)=54.21; P<0.0001). IL-1α reduced uptake in CTRLs, while IL-1β increased uptake; neither significantly affected SOD1 astrocytes, despite increased expression of both cytokines. Conclusion: ALS astrocytes exhibit impaired glutamate uptake and altered responsiveness to inflammatory stimuli, highlighting EAAT2 and inflammatory signaling as potential therapeutic targets. Funding: FAPERJ, CNPq, CAPES.