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

To enhance understanding of early diagnosis, differential disease progression rates, and the molecular profiles of human prion strains, we analyzed prion seeding activity over time in Creutzfeldt-Jakob disease (CJD)-infected mice using the real-time quaking-induced conversion (RT-QuIC) assay. Our previous work highlighted pre- clinical alterations in endocytic machinery and cytoskeleton-associated responses in CJD-affected brain regions. In this study, infectious prion strains derived from human CJD-MM1 and VV2 brain tissues were inoculated into tg340, tg361 (expressing approximately four times the human PrP-M129 and PrP-V129, respectively), and tg650 (expressing approximately six times the human PrP-M129) mice. A total of 188 brain samples (cortex) were analyzed from confirmed CJD-infected mice and control mice at both pre-clinical and clinical stages of the disease. Notably, we observed region- specific and PrP strain-specific differences in seeding activity at the pre-clinical stage of disease in CJD-MM1 and VV2 infected mice. The lag phase between the positive response ranged from 7.5 to 24.5 hours across all regions and disease stages. In the cortex, CJD-MM1-infected tg340 mice exhibited a prolonged lag phase ([~]24.5 hours), while CJD-VV2-infected tg341 mice showed minimal seeding response and relative fluorescence unit (RFU) signal rates. In contrast, the cerebellum of VV2 clinical stage mice exhibited a shorter lag phase, and VV2 preclinical stage mice showed significantly lower RFU signal rates. Proteomic profiling via SWATH-MS identified 500 and 682 differentially expressed proteins in the MM1 and VV2 models, respectively. Key proteins such as Gnl1, Stxbp1, Pllp, Gps1, Nefh, Ahsa1, Rala, Cacybp, Pdk1, Unc13a, Rab21, Rraga, Ppp1r9a, Eif4b, Atp2b2, Vps51, H2afx in CJD-MM1 and Gm10358, Tnc, Calb2, Ppm1h, Dnaja1, Gm45808, Hpcal1, Prkca, Dock3, Syn2, Agap2, Tmem126a, Fdps, Ndufa4 in CJD- VV2 showed significant alterations at the early pre-clinical stages, correlating with detectable prion replication. These molecular shifts highlight potential early-stage diagnostic biomarkers. Functional analyses revealed that both MM1 and VV2 subtypes engage early compensatory responses; however, MM1 primarily involves metabolic reprogramming and enhanced vesicle clearance, while VV2 is characterized by pronounced disturbances in calcium signaling and structural integrity at early stage of the disease. These findings emphasize the utility of RT-QuIC and proteomics in characterizing prion seeding and progression, providing valuable insights into the molecular mechanisms underlying prion diseases and potential early diagnostic markers.