Speaker Dr Szymon Manka, MRC Prion Unit and Institute of Prion Diseases, UCL

Title Spying on prion pathogenicity mechanisms in real time and across scales in cultured cells

Host Joe Atherton

 

Abstract Prions are protein-only infectious agents that cause invariably fatal neurodegeneration. Prototypic prions exhibit distinct amyloid fibril structures (strains) formed from misfolded prion protein (PrP) chains. It is unclear how prion accumulation in the brain causes rapid dementia. To study prion pathogenicity, we used cryo-correlative light and electron microscopy/tomography (cryo-CLEM/ET). Prion fibrils from terminally infected mice were tagged with fluorophores and used to inoculate primary neuronal and glial cultures. Growth and spread of PrP assemblies were tracked for up to 3 weeks using anti-PrP antibodies. We observed rapid internalisation of prion inocula by neurons, followed by progressive PrP aggregate formation. The infected neurons also showed interwoven sheet-like aggregates of uncertain origin and signs of mitochondrial pathology akin to those recently reported for an analogous in vitro model of Huntington's disease. To gain further insights into prion propagation dynamics, we have engineered neuroblastoma cells with an expanded genetic code to enable site-specific incorporation of a fluorophore-taggable non-canonical amino acid (ncAA) into PrP. Our fluorescent PrP appears to convert into fluorescent prions upon infection with ex vivo prion seeds, supporting live monitoring of prion infection. Using this system, we plan to identify key stages of prion propagation and analyse them structurally with cryo-correlative light, electron, and X-ray microscopy (cryo-CLEXM) to obtain multiscale mechanistic insights into prion pathogenicity mechanisms.