An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrP ^C ) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrP ^C proteoforms have been hampered by the lack of methods able to identify all PrP ^C -derived proteoforms. Building on previous knowledge of PrP ^C endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrP ^C constitutive processing and the relative abundance of PrP ^C proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrP ^C proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrP ^C -derived fragments. Endoproteolytic processing of PrP ^C was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrP ^C was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrP ^C is not affected by PrP ^C overexpression or host factors other than PrP ^C but can be impacted by PrP ^C primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrP ^C and for a comprehensive appraisal of PrP ^C proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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