Reversal of Mitochondrial Benefits, Depolarization of Mitochondria in Large Extracellular Vesicle, and Multi-Omics Remodeling After EGCG Plus Omega-3 Withdrawal in Children with Down Syndrome

Down syndrome (DS) is characterized by chronic oxidative stress, mitochondrial dysfunction and metabolic alterations contributing to neurodevelopmental impairment and accelerated aging. We previously showed that supplementation with epigallocatechin-3-gallate (EGCG) plus omega-3 fatty acids improved mitochondrial respiratory chain function in children with DS. Here, we investigated the bioenergetic and molecular consequences of a three-month withdrawal period following chronic supplementation of EGCG plus omega-3 in children with DS aged 6–17 years. Mitochondrial respiratory chain activities in peripheral blood mononuclear cells (n = 8) and mitochondrial content and polarization in large circulating extracellular vesicles (lEVs) (n = 4) were assessed at different time points. Whole-blood RNA sequencing and untargeted plasma metabolomics were performed at the end of supplementation (T0, n = 3) and after three months of withdrawal (T3, n = 4). Treatment discontinuation resulted in progressive loss of the previously observed bioenergetic benefits and the proportion of depolarized mitochondria in circulating lEVs increased at T3. Transcriptomic analysis revealed downregulation of pathways related to oxidative phosphorylation, mitophagy, autophagy, and lysosomal function. Metabolomic profiling showed remodeling of lipid and fatty-acid metabolism, consistent with impaired mitochondrial and redox homeostasis. These findings suggest that the mitochondrial benefits associated with nutraceutical supplementation are not sustained after treatment withdrawal and identify changes in circulating lEV-associated mitochondrial polarization as a potentially informative readout of mitochondrial homeostasis in DS.