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

The prion protein (PrPC) 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 compre-hensive investigations on the full spectrum of PrPC proteo-forms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent iden-tification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC- derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with alpha-cleavage-derived C1 repre-senting the most abundant proteoform and ADAM10-medi-ated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the pro-gression of neurodegenerative diseases.

Automated summary

The PrPC protein undergoes various endoproteolytic events, resulting in bioactive fragments with physiological functions and implications in neurodegenerative diseases. Lack of methods to identify all PrPC-derived proteoforms has hindered comprehensive investigations. Researchers developed an optimized Western blot assay based on previous knowledge, enabling the identification of the full spectrum of PrPC proteoforms in brain samples.