Cryo-ET reveals subcellular pathology in prion-infected neurons

Introduction: Prion infection is driven by the pathogen’s unique ability to associate with membranous environments, enabling efficient colonisation of both intra- and extracellular niches in neurons. However, detailed investigation into prion propagation within these subcellular microenvironments has remained largely inaccessible using conventional imaging techniques. To address this gap, we employed cryo-electron tomography (cryo-ET) to visualize prion-infected neurons in 3D and near-native states, revealing nanoscale structural features associated with infection.

Objectives:

• Identify sites of prion infection in neurons by cryo-correlative light and electron microscopy (cryo-CLEM), and image with cryo-ET.
• Reconstruct high resolution tomograms and segment infection-associated features for analysis.

Methods: Primary mouse neurons were cultured on cryo-EM grids and exposed to fluorophore-tagged ex vivo prions. Nascent PrP assemblies developed after 3 weeks and were immunolabelled live before vitrification, enabling precise localisation of both the original inoculum and nascent assemblies using cryo-CLEM. Cryo-ET of infection sites allowed high-resolution reconstruction of infected cellular microenvironments, followed by detailed 3D annotation by convolutional neural networks (CNNs).

Results: Short, highly decorated nascent prion fibrils were found intra- and extracellularly around neurites. Electron-dense sheets of unknown nature appear to bind and disrupt membranes in the vicinity of nascent prion fibrils. We begin to infer staging of this newly uncovered sheet aggregate formation, which may be an important feature of prion infection at the subcellular level. 

Conclusions:
Cryo-ET and CNN-based analysis of vitrified, prion-infected neurons reveal membrane-associated ultrastructural changes and electron-dense sheet aggregates as potential contributors to prion pathology. Ongoing work aims to define their origin and composition.