Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures
Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures
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DissertationDate of Exam
29.11.2018Date of Publication
19.02.2019Advisor
Vorberg, InaCo-Referee
Höhfeld, JörgMetadata
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Abstract
Prions are unconventional infectious agents that cause always fatal neurodegenerative diseases termed prion disease or transmissible spongiform encephalopathies in mammals. Prion diseases are caused by an accumulation of the misfolded, aggregated host encoded prion protein (PrP). The normal, cellular, α-helix rich isoform (PrPC) is converted into the disease-associated β-sheet rich pathogenic isoform (PrPSc). PrPSc can adopt multiple conformations that likely encipher prion strain characteristics. Currently, prion therapeutic clinical trials lack success and there is an urgent need for novel therapeutics. The aim of this study was to develop a cell-based assay for high content screening of large compound libraries with an automated microscope to identify compounds that might impair prion replication. Furthermore, identified compounds should be tested on prion infected organotypic slice cultures to test whether in vitro detected anti-prion compounds are also effective in a more complex neuronal environment. Additionally, two promising compounds, FeTMPyP and PIM-B31, identified by our collaboration partner Emiliano Biasini (University of Trento), were tested ex vivo. Beside this a comparative study of host response between ex vivo and in vivo should evaluate the transferability between the two systems, as this has not been was not shown until now. In the established screen 152 compounds were tested, 84 had an inhibitory effect on PrPSc accumulation in persistently infected N2a22L cells and the seven strongest inhibitors were further validated by western blot analysis. The most promising candidate, PHA665752, was tested ex vivo and showed a reduction of PrPSc accumulation that was however not significant. FeTMPyP showed strong toxicity and PIM-B31 showed inconsistent results that depended on different concentration and strain-specificity. Beside this, pathway analysis of ex vivo and in vivo infected mouse cerebella with different strains at various time points was performed with DAVID 6.8, an online bioinformatics resource. Analysis of the 250 most significant differentially expressed genes revealed that several comparable pathways were changed due to prion infection in brain slices and brains. The calcium signaling pathways and neuroactive ligand-receptor pathways were deregulated the most by prion infection ex vivo as well as in vivo.
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570 Biowissenschaften, BiologieZielinski, Catharina: Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures. - Bonn, 2019. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-53053
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-53053
@phdthesis{handle:20.500.11811/7833,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-53053,
author = {{Catharina Zielinski}},
title = {Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2019,
month = feb,
note = {Prions are unconventional infectious agents that cause always fatal neurodegenerative diseases termed prion disease or transmissible spongiform encephalopathies in mammals. Prion diseases are caused by an accumulation of the misfolded, aggregated host encoded prion protein (PrP). The normal, cellular, α-helix rich isoform (PrPC) is converted into the disease-associated β-sheet rich pathogenic isoform (PrPSc). PrPSc can adopt multiple conformations that likely encipher prion strain characteristics. Currently, prion therapeutic clinical trials lack success and there is an urgent need for novel therapeutics. The aim of this study was to develop a cell-based assay for high content screening of large compound libraries with an automated microscope to identify compounds that might impair prion replication. Furthermore, identified compounds should be tested on prion infected organotypic slice cultures to test whether in vitro detected anti-prion compounds are also effective in a more complex neuronal environment. Additionally, two promising compounds, FeTMPyP and PIM-B31, identified by our collaboration partner Emiliano Biasini (University of Trento), were tested ex vivo. Beside this a comparative study of host response between ex vivo and in vivo should evaluate the transferability between the two systems, as this has not been was not shown until now. In the established screen 152 compounds were tested, 84 had an inhibitory effect on PrPSc accumulation in persistently infected N2a22L cells and the seven strongest inhibitors were further validated by western blot analysis. The most promising candidate, PHA665752, was tested ex vivo and showed a reduction of PrPSc accumulation that was however not significant. FeTMPyP showed strong toxicity and PIM-B31 showed inconsistent results that depended on different concentration and strain-specificity. Beside this, pathway analysis of ex vivo and in vivo infected mouse cerebella with different strains at various time points was performed with DAVID 6.8, an online bioinformatics resource. Analysis of the 250 most significant differentially expressed genes revealed that several comparable pathways were changed due to prion infection in brain slices and brains. The calcium signaling pathways and neuroactive ligand-receptor pathways were deregulated the most by prion infection ex vivo as well as in vivo.},
url = {https://hdl.handle.net/20.500.11811/7833}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-53053,
author = {{Catharina Zielinski}},
title = {Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2019,
month = feb,
note = {Prions are unconventional infectious agents that cause always fatal neurodegenerative diseases termed prion disease or transmissible spongiform encephalopathies in mammals. Prion diseases are caused by an accumulation of the misfolded, aggregated host encoded prion protein (PrP). The normal, cellular, α-helix rich isoform (PrPC) is converted into the disease-associated β-sheet rich pathogenic isoform (PrPSc). PrPSc can adopt multiple conformations that likely encipher prion strain characteristics. Currently, prion therapeutic clinical trials lack success and there is an urgent need for novel therapeutics. The aim of this study was to develop a cell-based assay for high content screening of large compound libraries with an automated microscope to identify compounds that might impair prion replication. Furthermore, identified compounds should be tested on prion infected organotypic slice cultures to test whether in vitro detected anti-prion compounds are also effective in a more complex neuronal environment. Additionally, two promising compounds, FeTMPyP and PIM-B31, identified by our collaboration partner Emiliano Biasini (University of Trento), were tested ex vivo. Beside this a comparative study of host response between ex vivo and in vivo should evaluate the transferability between the two systems, as this has not been was not shown until now. In the established screen 152 compounds were tested, 84 had an inhibitory effect on PrPSc accumulation in persistently infected N2a22L cells and the seven strongest inhibitors were further validated by western blot analysis. The most promising candidate, PHA665752, was tested ex vivo and showed a reduction of PrPSc accumulation that was however not significant. FeTMPyP showed strong toxicity and PIM-B31 showed inconsistent results that depended on different concentration and strain-specificity. Beside this, pathway analysis of ex vivo and in vivo infected mouse cerebella with different strains at various time points was performed with DAVID 6.8, an online bioinformatics resource. Analysis of the 250 most significant differentially expressed genes revealed that several comparable pathways were changed due to prion infection in brain slices and brains. The calcium signaling pathways and neuroactive ligand-receptor pathways were deregulated the most by prion infection ex vivo as well as in vivo.},
url = {https://hdl.handle.net/20.500.11811/7833}
}
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