Periodic Reporting for period 1 - Prionomics (Systematic profiling of molecular changes during prion disease progression)
Reporting period: 2016-03-01 to 2018-02-28
Prion diseases are fatal neurodegenerative diseases and are caused by proteinaceous infectious particles termed prions. The infectious agent of prion diseases has been identified decades ago, but the actual cellular processes that subsequently cause neurons to degenerate remain poorly understood. While the mechanisms leading to neurodegeneration are largely unknown, several molecular pathways have been implicated to play a role. Interestingly, the same pathways as well as protein aggregation, have also been linked to other neurodegenerative diseases including Alzheimer’s Disease and Parkinson’s Disease. While these neurodegenerative diseases have a distinct pathology, they might share a common mechanism of protein misfolding induced neuronal death. This is particularly interesting as the prion mouse model recapitulates the human disease more faithfully than other neurodegenerative disease mouse models. With more than 50 million people suffering from neurodegenerative diseases worldwide, and no treatment options being available that prevent or even just slow down their progression, neurodegenerative diseases represent a major burden for patients, their families as well as our society. By advancing our understanding of neurodegenerative diseases, this project has thus a major impact on our society.
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.
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.
Objective 1: Profiling of transcriptional and translational changes during prion disease progression
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.
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.
Neurodegenerative diseases affect more than 50 million people worldwide, and, due to our demographic development, their prevalence is steadily rising. Neurodegenerative diseases are devastating disorders, and no therapeutics are currently available that can cure or even just slow down their progression. Thus, patients, their families as well as our society are in urgent need of novel and better therapeutics.
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.
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.