Study of new diagnostic markers in protein misfolding diseases

Neurodegenerative diseases caused by protein misfolding, such as prionopathies and synucleinopathies, still lack reliable, minimally invasive diagnostic biomarkers. Seed amplification assays (SAAs)—a class of ultrasensitive techniques that exploit the self-propagating nature of misfolded protein aggregates to amplify and detect minute amounts of pathological seeds—have emerged as powerful tools for identifying disease-specific protein aggregates in easily accessible biological samples. Among these, the real-time quaking-induced conversion (RT-QuIC) technique has proven to be one of the most robust and reproducible approaches. RT-QuIC is based on the principle that pathological protein seeds can induce the conformational conversion of recombinant normal proteins into misfolded aggregates, whose formation is stimulated by cycles of vigorous shaking at elevated temperatures and is continuously monitored through a fluorescence readout in real time. This doctoral research aimed to optimize RT-QuIC conditions for application in peripheral biofluids—specifically saliva and tear fluid (TF)—to assess their feasibility, analytical robustness, and diagnostic potential in Creutzfeldt–Jakob disease (CJD) and Parkinson’s disease (PD). Particular attention was given to pre-analytical variables related to sample handling and preparation, such as freeze–thaw cycles, storage conditions, sonication, and sampling protocols, which were shown to critically influence assay reproducibility. Moreover, the choice of recombinant protein substrate proved pivotal, with different performances observed among prion proteins from diverse mammalian species and commercially standardized α-synuclein (α-syn) showing improved inter-laboratory reproducibility compared to in-house preparations. The optimized assays demonstrated that RT-QuIC performed on saliva and TF can discriminate CJD and PD patients from controls. In addition, for CJD, where paired cerebrospinal fluid (CSF) samples were available, RT-QuIC results obtained from saliva and TF were compared with those from CSF, revealing lower sensitivity in peripheral fluids but confirming a consistent diagnostic pattern. This comparison was not performed for PD, due to the lack of corresponding CSF samples. Despite these limitations, saliva and TF offer clear advantages in terms of non-invasiveness and suitability for longitudinal monitoring.These findings confirm saliva and TF as promising alternative matrices for RT-QuIC–based detection of pathological prion protein (PrPSc) and α-syn seeds, and highlight the need for harmonization of protocols, quantitative adaptations, and integration with complementary biomarkers such as neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and advanced imaging techniques. In conclusion, this work contributes to the development of minimally invasive, standardized, and clinically relevant diagnostic tools, laying the groundwork for future applications in early detection, patient stratification, and therapeutic monitoring in protein misfolding diseases.