Cell-type and strain-resolved analysis of prion disease progression integrating FANS/RNA-seq, prion titre quantification, and proteomics

Advances in cryo-electron microscopy have refined our understanding of prion strains, establishing that strain identity is conformationally encoded rather than defined solely by histopathology, biochemistry, or incubation time. Yet it remains unclear how structurally distinct assemblies of the same host-encoded prion protein elicit divergent neuropathological phenotypes. In particular, the cell-type-specific responses of neurons and glia to different prion strains are poorly characterised.

To address this, we developed an optimised protocol combining four-way fluorescence-activated nuclear sorting (FANS) with RNA sequencing to enable high-purity transcriptional profiling of neurons, astrocytes, oligodendrocytes, and microglia from individual brain samples. Mice were intracerebrally inoculated with structurally distinct murine-adapted prion strains (RML, ME7, and 22L) and sampled at multiple stages of disease, spanning early exponential propagation to neurodegeneration endpoints. From the same samples, we quantified prion titres using the Scrapie cell assay and performed mass spectrometry-based proteomic analyses.

This integrative approach situates transcriptional dynamics within a well-defined temporal and molecular framework of prion infection, allowing direct, multimodal comparisons of strain- and cell-type-specific responses. We will present novel insights into the mechanisms by which prion strains differentially engage CNS cell types across disease progression.