Prion diseases are rare neurodegenerative disorders that are deadly, incurable, and caused by misfolded prion proteins (PrPSc) in the brain leading to neuropathological changes and rapid cognitive decline. The paucity of current approaches to prion disease research has hampered progress and so here we address two technical challenges that have limited research tools. Firstly, human prion isolates are incompatible with most cellular prion infection models, limiting their clinical relevance. Secondly, genome-wide transcriptional studies of bulk brain tissue cannot resolve the contribution of different brain cell types to prion pathogenesis. This makes it difficult to understand the relationship between prion accumulation, pathogenesis, and neurotoxicity. In the first part of this thesis we set out to develop novel in vitro cellular models of human prion infection using two approaches: (a) the prion organotypic slice culture assay (POSCA) and (b) an immortalized human neural progenitor cell line (ReN). In these two experimental paradigms, prion replication was sensitively tracked over several weeks through measurements of amyloid seeding activity with real time quaking induced conversion (RT-QuIC). Deer mouse cerebellar slice cultures and PrPC-overexpressing ReN cultures were found to resist infection with human prion isolates. While we were unable to establish an in vitro model of human prion infection, our findings reflect the inherent difficulty of establishing clinically relevant cellular models. In the second part of this thesis, we compared prion-elicited transcriptional responses between mouse brain cell types to shed further insight into prion neuropathology. Using bulk RNA sequencing, gene expression was examined longitudinally in microdissected brain tissues from an in vivo mouse model of RML Scrapie infection. We then applied single-cell RNA sequencing to brain cells isolated at the clinical endpoint of RML Scrapie infection. A single-cell atlas of nearly 150,000 high-quality transcriptomes was constructed that permitted characterization of disease-associated transcription within individual brain cells. Analysis of these two transcriptional datasets identified widespread prion-altered molecules and pathways across brain cell types and revealed a new layer of resolution to prion pathobiology.