TETRAPLOID ASTROCYTES PRESENT FUNCTIONAL CALCIUM ACTIVITY IN PRIMARY CULTURES FROM DRUG-RESISTANT EPILEPSY PATIENTS

Drug-resistant epilepsy (DRE) affects 25–33% of patients who do not respond to current antiseizure treatments. However, the exact etiology of DRE has not been fully characterized. We have recently identified tetraploid astrocytes, characterized by a double DNA content equivalent to 4C, in the brain of subjects without diseased affecting the central nervous system. Tetraploid astrocytes are significantly increased in the epileptogenic zone of patients with DRE. However, functional properties of tetraploid astrocytes have not been characterized yet. This study aimed to assess whether tetraploid astrocytes are functionally active. We used intracellular calcium [Ca²⁺]ᵢ imaging to assess astrocyte function in two models: primary astrocyte cultures derived from resected cortical tissue of DRE patients and a differentiated human astrocyte cell line (CRL-8621). Membrane receptor functionality was evaluated by measuring Ca²⁺ responses to various stimuli (K⁺, bradykinin, ATP, and Bz-ATP). Astrocytic tetraploidy was evaluated using DAPI staining and ImageJ. Tetraploid astrocytes from patients with DRE showed functional calcium responses to potassium chloride (KCl), adenosine triphosphate (ATP), which were partially mediated by P2X7 receptor activation, as evidenced by responses to the selective agonist Bz-ATP. Similarly, tetraploid astrocytes identified in line cell exhibited functional responses to ATP, including P2X7-dependent signaling, and bradykinin. These results demonstrate for the first time that tetraploid astrocytes are a functionally active population comparable to diploid astrocytes using two different in vitro models. These findings may contribute to identifying new therapeutic targets of DRE in the future.