Proteomic and Kinetic Characterization of Prion Seeding in Distinct Human CJD Strains Unveils Early Diagnostic Biomarkers

To enhance understanding of early diagnosis, disease progression, and molecular signatures of human prion strains, we analyzed prion seeding activity in Creutzfeldt Jakob disease (CJD) infected mice using the real-time quaking-induced conversion (RT-QuIC) assay. Infectious prion strains derived from CJD-MM1 and VV2 human brain tissues were inoculated into tg340, tg361 (expressing ~4x human PrP-M129 or PrP-V129), and tg650 (expressing ~6x human PrP-M129) mice. We examined 188 brain samples from confirmed CJD-infected and control mice at pre-clinical and clinical stages. Region- and strain-specific differences in seeding activity emerged at pre-clinical stages. Lag phases ranged from 7.5 - 24.5 h across regions and stages: MM1-infected tg340 cortex displayed prolonged lag (~24.5 h), while VV2-infected tg341 cortex showed minimal seeding. In contrast, VV2 cerebellum exhibited shorter lag phases at clinical stage but significantly reduced fluorescence signals at pre-clinical stage. Proteomic profiling (SWATH-MS) revealed 500 and 682 differentially expressed proteins in MM1 and VV2 models, respectively. Early pre-clinical alterations included Gnl1, Stxbp1, Pllp, Nefh, Ahsa1, Rala, and Atp2b2 in MM1, and Tnc, Calb2, Dnaja1, Hpcal1, Prkca, and Syn2 in VV2, correlating with detectable prion replication. Functional analyses suggested distinct compensatory responses: MM1 involved metabolic reprogramming and vesicle clearance, while VV2 showed early calcium signaling disturbances and structural impairment. These findings demonstrate that RT-QuIC combined with proteomics enables sensitive detection of strain-specific pre-clinical changes, providing mechanistic insights into prion pathogenesis and identifying potential biomarkers for early diagnosis.