TNFa-NF-?B activation through pathological a-Synuclein disrupts the BBB and exacerbates axonopathy

Dysfunction of the blood brain barrier (BBB) is recognized as a key factor in the progression of neurodegenerative diseases, but the detailed mechanisms behind its pathogenesis and impact on neurodegeneration remain elusive. This study aimed to reveal the pathological effects of a-Synuclein (a-Syn), an aggregation protein in Synucleinopathy, on BBB integrity and function, and identify therapeutic targets for a-Syn-related vasculopathy. Using a brain endothelial cell model, we investigated the pathological effect of preformed fibril a-Syn (PFF) on BBB integrity, employing generative adversarial network (GAN) deep learning to analyze pathological changes. We found that PFF activates immune responses, increasing endothelial monolayer permeability via the TNFa-NF-?B pathway. Further in vivo studies with PFF induced a-Synucleinopathy and transgenic animal model (G2-3) revealed that a-Syn aggregation, disrupts the BBB, leading to axonal degeneration that was mitigated by treatment with a non-BBB-penetrating TNFa inhibitor, Etanercept. These findings suggest that targeting brain endothelial TNFa signaling could be a potential therapeutic approach for Synucleinopathy-related NDs. Overall design: Primary human brain microvascular endothelial cells (HBMVECs) were cultured and treated with control, monomeric a-synuclein, or pre-formed fibrils (PFF) of a-synuclein for 24 or 72 hours. After treatment, RNA was extracted for transcriptomic analysis to investigate gene expression changes induced by different forms of a-synuclein.