Prion detection in peripheral tissues and biofluids of patients at different stages of sporadic and genetic prion diseases

Prion diseases (PrDs) are fatal neurodegenerative disorders caused by the misfolding of the cellular prion protein (PrPC) into its pathological isoform (PrPSc). Despite major advances in understanding their molecular mechanisms, definitive diagnosis still relies largely on post-mortem neuropathological confirmation. Ultrasensitive diagnostic techniques have emerged in the context of PrDs as a revolutionary diagnostic tool, allowing the detection of PrPSc in CSF and other easily accessible tissues. The sensitive detection of prions in peripheral tissues and fluids during life, however, remains an unmet challenge. This thesis aimed to optimize and apply the Protein Misfolding Cyclic Amplification (PMCA) technique for the detection of PrPSc in peripheral biological matrices of both sporadic and genetic PrDs, with a particular focus on sporadic Creutzfeldt-Jakob disease (sCJD) and Fatal Familial Insomnia (FFI). In the first part, PMCA was systematically optimized for use with olfactory mucosa (OM) samples obtained from sCJD patients. The developed assay achieved high diagnostic sensitivity and absolute specificity, allowing for the reliable amplification of PrPSc from non-invasive nasal brushings. These findings demonstrated that prion seeds can be detected in an accessible tissue OM, and validated PMCA as a robust diagnostic tool in sCJD. Building upon this methodological foundation, the second part of the thesis extended PMCA analyses to longitudinal biological samples collected from patients with FFI at different stages of the disease. Through the use of optimized substrates and reaction conditions, PrPSc was detected in blood and urine samples of symptomatic patients and in a relatively high proportion even in presymptomatic patients, carriers of the pathological mutation. The inclusion of samples from the DOXIFF clinical trial, alongside untreated Spanish mutation carriers, provided unique insight into disease progression, treatment effects, and the temporal dynamics of prion detection in peripheral biofluids. Overall, this work demonstrates the applicability of PMCA as a powerful ultrasensitive assay for detecting prions in minimally invasive specimens. By expanding prion detection beyond the central nervous system, the findings contribute to the development of early diagnostic and monitoring tools for both sporadic and genetic PrDs.