Cellular mechanisms of Prion Propagation

Prions, the infectious proteins that cause prion diseases have a unique way of reproducing themselves. This process was originally thought to be specific only to prions, but now we know that similar self-replicating mechanisms are found in other proteins associated with diseases like Alzheimer's and Parkinson's. Understanding how these proteins replicate is crucial for assessing risks and for developing treatments. To do this, we are using advanced technologies and genetic tools to study the molecular processes involved in the replication of these disease-related proteins. Our goal is to learn more about how prions replicate in brain cells. We want to understand the specific ways these proteins assemble, investigate how they cause harm, figure out how the abnormal forms of these proteins are directed to different parts of the cell, and investigate how they spread in brains of mice during the early stages of disease. To achieve this, we use a combination of advanced imaging techniques, genetic engineering to create special tools, and in-depth molecular analyses.

Self-templating prion replication is a fundamental aspect of prion pathogenesis, exemplifying a prototypic non-nucleic acid-based mechanism of infection and heritability. Initially believed exclusive to prions, proteopathic seed self-assembly is now recognized as a shared trait among amyloids in neurodegenerative disorders like Alzheimer’s and Parkinson’s diseases. Identifying shared and distinct disease pathways is crucial for evaluating risks and developing therapeutic strategies. Gaining a deeper understanding of the molecular mechanism is thus essential to elucidate both specific and shared disease pathways.

We aim to advance our understanding of prion replication by investigating the molecular basis of protein self-assembly, explore the pathogenicity of PrP amyloids, decipher how abnormal PrP is sorted into secretory pathways and monitor how prions colonise mouse brains at early stages of disease. Employing an interdisciplinary approach, we utilise advanced imaging technologies, genetically engineer biomolecular tools, and conduct comprehensive molecular analyses to address self-templating protein aggregation. The specific objectives of this program are detailed below.

The plasma membrane is the first site of PrP conversion, but the molecular players that facilitate this process are largely unknown. We will explore the role of membrane microdomains in abnormal PrP formation and examine the link between PrP conversion and canonical/non-canonical uptake pathways. Following identification of key mediators of endocytosis, we will further investigate their functional protein interaction networks to validate their role in PrP conversion.

We aim to utilise 3D brain imaging to better characterise early changes in neurodegeneration. Whole mouse brain imaging with high resolution and ultra-fast processing times will be conducted with lightsheet and label-free block-face serial section microscopy. This advanced imaging approach will be integrated into a machine-learning assisted imaging analysis pipeline with anatomical registration to the Allen brain atlas.

While amyloid deposits are a shared feature of all neurodegenerative diseases, it remains an ongoing debate whether they are inert or actively contribute to neuronal demise. We established a model for amyloid assemblies based on fibrillar aggregate formation at the extracellular matrix of prion-infected cells. After decellularisation, this cell-free amyloid model facilitates the study of its infectious and toxic characteristics when plated with neuronal or primary neuronal cells. Our aims include, but are not restricted to investigating the role of proteinases in the mobilisation of amyloid fibrils and toxic endpoints.

We further aim to delineate the cellular trafficking pathways of both normal and abnormal PrP, including their segregation into exocytic pathways. We have pinpointed the octapeptide region of Prnp as a viable target region for generating chimeras while sustaining PrP conversion.