Periodic Reporting for period 1 - Prionomics (Systematic profiling of molecular changes during prion disease progression)
Reporting period: 2016-03-01 to 2018-02-28
The molecular underpinnings of prion disease pathology are beginning to get unraveled, yet a detailed mechanistic understanding of the deleterious effects of prions is missing. A thorough analysis of the molecular changes during disease progression is therefore key to developing a deeper understanding of how prions escape immune surveillance and eventually lead to neuronal cell death. Curiously, few tissues have been shown to replicate prions, and prions induce cell loss, plaque formation and vacuole accumulation only in the brain. Moreover, some cell types can clear prions under certain conditions, suggesting that the expression of co-factors regulates whether prions are cleared, replicate or exert toxicity. While it is currently unknown why different cell types undergo distinct fates upon prion infection, these observations emphasize the importance of studying prion disease in a cell-type specific manner.
The proposed project entailed the systematic and unbiased analysis of genome-wide cell-type specific molecular changes during prion disease pathogenesis in vivo. The assessment of multiple time points following prion infection allows the dissection of the dynamics of disease manifestation. Furthermore, focusing specifically on cells that are relevant to prion disease, sheds light on the role and interplay of the analyzed cell types. The thereby generated data yields novel and important insights into prion disease pathophysiology, and additionally contributes to our understanding of other neurodegenerative diseases.
To overcome limitations associated with analyzing entire tissues, novel technologies such as cell-type specific ribosome profiling have been implemented. Furthermore, the implementation of such technologies made whole-tissue transcriptome profiling obsolete. Multiple transgenic mouse strains that express GFP tagged ribosome specifically in cell types that are relevant to prion disease have been generated and required protocols were successfully adapted and optimized. Mice were injected with non-infectious and prion-infected brain homogenates and sacrificed at multiple times points during the disease as well as the terminal stage of the disease. All mice showed well-documented and characteristic clinical symptoms and expressed GFP-tagged ribosomes only in the desired cell types. Brain samples were subsequently collected and frozen for both immunohistochemical and biochemical analysis. 90 cell-type specific ribosome profiling have been generated and submitted for high-throughput sequencing after passing quality control checkpoints.
Objective 2: Identification of candidate genes important for prion pathophysiology
A thorough bioinformatic analysis is key to interpreting genome-wide data and a customized pipeline was set up to analyze cell-type specific ribosome profiling datasets. 40 datasets have successfully been analyzed and identified cell-type specific molecular changes during prion disease. In parallel, immunohistochemical analysis of all time points confirmed the appearance of typical hallmarks of prion disease.
Objective 3: Validate the importance of specific molecular changes for pathophysiology
Brain samples from all transgenic mouse strains and all time points have been frozen. Immunohistochemial analysis confirmed the presence of disease hallmarks, allowing the samples to be used for the validation of cell-type specific molecular changes.
The initial proposal was adapted to incorporate novel technologies, allowing the identification of cell-type specific molecular changes during prion disease progression. The thereby generated data will yield novel insights at a higher resolution, which will lead to a better understanding of neurodegenerative diseases and will have a higher societal impact than the originally anticipated data. One drawback of the novel technologies was the requirement of additional tools and a re-definition of initial work packages. Consequently, not all tasks have been fully implemented to date. As soon as the final work package is completed, all data will be published in open-access peer-reviewed scientific journals, presented to a broad scientific audience at conferences and meetings, and will be made publicly available on a website, acknowledging the EU’s contribution.
Identifying a comprehensive list of molecular changes associated with different stages of prion disease as well as other neurodegenerative diseases, is a major step towards developing a better understanding of these diseases. Furthermore, the analysis of specific disease-relevant cell types at multiple time points, allows the identification of genes that are important for different aspects of disease pathogenesis.
The project's outcome not only contributes to a better understanding of prion diseases and neurodegenerative diseases but also provide potential starting points for the development of targeted therapeutic interventions, and thus have a major societal impact.