PHOSPHATIDIC ACID-MEDIATED PRION AGGREGATES DISRUPT MITOCHONDRIAL ACTIVITY

The cellular prion protein (PrP^C), highly expressed in neurons, participates in various signaling pathways. Its misfolding into the pathogenic prion scrapie (PrP^Sc) results in amyloid aggregate formation, a hallmark of prion diseases. Mitochondrial dysfunction is central to neurodegenerative pathogenesis, and oligomeric species are often more toxic than mature fibrils. The mechanisms linking PrP aggregation to mitochondrial impairment, however, remain unclear. The objective of the present study is to investigate the effects of phosphatidic acid (PA)-induced prion aggregates on mitochondrial function and cell viability in human neuroblastoma cells (SH-SY5Y cell line). We exposed SH-SY5Y cells to intact or fragmented PrP:PA amyloid fibrils for 48 hours. Cytotoxicity was assessed via lactate dehydrogenase (LDH) assay. The morphology of PrP:PA aggregates was observed by transmission electron microscopy (TEM). Mitochondrial respiration was measured using high-resolution respirometry, and mitochondrial content by citrate synthase activity. Expression of respiratory complexes was evaluated by Western blotting, and the cellular distribution of PrP^C and PrP:PA fibrils by confocal microscopy. Cellular ROS was assessed using the DCFDA probe. We found that fragmented PrP:PA fibrils caused approximately 40% cell death in cultured cells, while intact fibrils were non-toxic. Cells exposed to fragmented PrP:PA aggregates showed increased complex I-mediated respiration, likely due to elevated expression of respiratory complexes, with no significant change in mitochondrial content. Preliminary data suggest that the increase in cellular respiration by PrP:PA aggregates drives ROS generation. Confocal imaging indicated preserved PrP^C membrane localization but revealed nuclear interaction of fragmented aggregates, unlike intact fibrils. Our findings suggest that fragmented PrP:PA aggregates promote cytotoxicity through mitochondrial modulation and nuclear interaction, unlike their intact counterparts. These findings highlight the pathological potential of prion aggregate fragmentation and support further investigation into lipid-mediated prion toxicity mechanisms.

This work was supported by Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ), Conselho Nac. Des. Cient. Tecnologico (CNPq) and CAPES.