Ratio shift of protein in braincells causes changes underlying early cognitivedecline SciReports
flasks, using 1.2–2 μg of each channel subunit plasmid and 0.4 μg of plasmid encoding eGFP in 8 mls of DMEM. Experiments to resolve the effects of co-expression of a 4R-tau isoform were accomplished by using 2 μg of construct in addition to those listed above. Cells were seeded into 35 mm culture dishes 24 h after transfection and cells expressing enhanced green fluorescent protein were used for electrophysiology 24 h later.
Samples were then washed with co-IP buffer four times before 60 μl Laemmli buffer was added to each condition. 12% polyacrylamide gels were used to separate proteins based on molecular weight by SDS-PAGE using the BioRad electrophoresis system. Gels were run for 1.5 h at a constant voltage of 150 V in running buffer . Wet transfer onto PVDF membranes was performed at 400 mA for 1 h in transfer buffer .
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Distinct translatome changes in specific neural populations precede electroencephalographic changes in prion-infected miceAuthor summary Prions are infectious agents composed of a misfolded protein. When isolated from a mammalian brain and transferred to the same host species, prions will cause the same neurodegenerative disease affecting the same brain regions and cell types. This concept of selective vulnerability is also a feature of more common types of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s. To better understand the mechanisms behind selective vulnerability, we studied disease responses of five cell types with different vulnerabilities in prion-infected mice at two different disease stages. Responses were measured as changes to mRNAs undergoing translation, referred to as the translatome. Before prion-infected mice demonstrated typical disease signs, electroencephalography (a method used clinically to characterize neurodegeneration in humans) revealed brain changes resembling those in human prion diseases, and surprisingly, the translatomes of all cells were drastically changed. Furthermore, before electroencephalography changes emerged, three cell types made unique responses while the most vulnerable cell type did not. These results suggests that mechanisms causing selective vulnerability will be difficult to dissect and that therapies will likely need to be provided before clinical signs emerge and individually engage multiple cell types and their distinct molecular pathways.
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