Prion Protein E200K Mutation Disrupts Human Neuronal Network Stability and Oxidative Stress Resilience

In mice, cellular prion protein (PrPC) has been shown to regulate neuronal network communication and protect neurons from oxidative stress damage. These roles are lost or corrupted when PrPC misfolds into pathogenic isoforms (prions) in prion disease. It remains unknown if PrPC plays similar roles in the human brain. Furthermore, prion protein structure is altered to some degree by an E200K mutation (PrPE200K) within its core residues. This autosomal-dominant mutation predisposes carriers to develop genetic Creutzfeldt-Jakob Disease (gCJD), the most common human genetic prion disease. Whether the mutation causes a loss or corruption of prion protein roles, leading to the disease, has not yet been determined. We hypothesize that the E200K mutation drives gCJD by exerting toxic effects that disrupt PrPC's normal activity, rendering neurons highly vulnerable to oxidative stress, neurotransmitter imbalance, cytoskeletal damage, and impaired vesicular transport. The synergic effect disrupts neural network connectivity. Here, we utilized a novel technology, a mesh multi-electrode array (MEA; MultiChannel Systems, Germany), to evaluate neuronal network function in human cerebral organoids (COs) expressing prion protein with and without the E200K mutation, or lacking prion protein. COs were generated from human induced pluripotent stem cells, cultured for at least 5 months, and adhered to the mesh MEA using a Polyethyleneimine solution and laminin. They were then cultured in BrainPhys media (StemCell) for at least a month, while monitoring the neuronal network activity. We found that a complete loss of PrPC increased neuronal activity due to impaired excitation regulation, predisposing organoids to oxidative stress-induced damage. COs heterozygous for PrPE200K showed reduced neuronal firing and connectivity, as well as enhanced susceptibility to oxidative stress-induced damage. This suggests that the PrPE200K was either neurotoxic or it corrupted the normal function of its co-expressed PrPC. Importantly, similar phenotypes were observed in COs homozygous for PrPE200K, suggesting that the mutation was toxic and increased COs' vulnerability to oxidative stress. Furthermore, proteomic and metabolomic analyses revealed that PrPE200K reduced neural motor transport, depleted GABA levels, and increased acetylcholine levels. Together, mesh MEA long-term monitoring of network activity in live COs, coupled with biochemical analyses, suggests that PrPE200K causes neuronal network breakdown associated with oxidative stress, vesicular transport, and neurotransmitter trafficking and use, distinct from PrPC loss-of-function. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this abstract are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.