Adult ADHD, PARK2 copy number variations and the blood-brain barrier: Culture duration-dependent metabolic alterations in human induced pluripotent stem cell-derived in vitro models

Background Attention-deficit/hyperactivity disorder (ADHD) is a multifactorial neurodevelopmental disorder in which PARK2 copy number variations (CNVs) constitute a rare genetic risk factor. PARK2 encodes parkin, a key regulator of mitochondrial quality control. Mitochondrial dysfunction is implicated in ADHD pathophysiology and can contribute to blood-brain barrier (BBB) disruption. As BBB disruption remains insufficiently understood in ADHD, this study investigated whether adult ADHD and PARK2 CNVs are accompanied by alterations in BBB phenotype, mitochondrial properties, and cellular metabolism. Methods Brain capillary endothelial-like cells (BCECs) were generated using 10 donor-specific human induced pluripotent stem cell (hiPSC) lines from adults with ADHD carrying PARK2 CNVs, alongside PARK2 wild type adults with and without ADHD. BCECs were analyzed at the end of differentiation (day 10) and after prolonged culture (day 17) to assess group-associated phenotypes and culture duration-dependent differences. Results Across all groups, BCECs showed brain endothelial marker expression, high transendothelial electrical resistance (TEER), and low sodium fluorescein (NaFl) permeability at day 10. After prolonged culture, NaFl permeability remained low, whereas TEER-based barrier integrity declined. Prolonged culture of BCECs reduced glycolytic ATP production rates while increasing mitochondrial ATP production and its relative contribution to total ATP production, reflecting a metabolic shift from glycolysis toward oxidative phosphorylation. Despite decreased total ATP production after prolonged culture, cellular energy status was largely preserved, alongside overall lower intracellular reactive oxygen species (ROS) and mitochondrial membrane potential. At day 10, BCECs from ADHD donors carrying PARK2 duplications displayed greater mitochondrial abundance, the highest basal and oxidative stress-induced intracellular ROS levels, and reduced TEER-based barrier integrity, while at day 17, BCECs from ADHD donors with PARK2 deletions exhibited the least glycolysis-dependent ATP production profile, although absolute mitochondrial ATP production remained comparable between groups. Conclusions Prolonged culture was associated with broad, ADHD- and PARK2 genotype-independent metabolic remodeling, whereas mitochondrial and metabolic alterations associated with ADHD and PARK2 CNVs were overall small. This study identifies culture duration as a critical parameter for the characterization of hiPSC-derived BBB in vitro models and supports the feasibility of using donor-specific BCECs at the end of differentiation to investigate BBB-associated mechanisms in adult ADHD and PARK2 CNVs.