Untersuchungen zu den selbst-replizierenden Eigenschaften des pathogenen Prion-Proteins beim Menschen

Abstract

Prion diseases are transmissible, fatal neurodegenerative diseases of humans and animals. They are linked to the conversion of the cellular prion protein (PrPC) into its pathogenic form (PrPSc). Due to this conversion prion diseases are pathogenic and transmissible. Until today, neither the underlying pathogenic mechanism is understood nor has a treatment been found. To make a definite diagnosis of a sporadic prion disease a brain biopsy is required. For this reason most often a probable prion disease is diagnosed in patient’s lifetime. In combination with the clinical heterogeneity newest findings suggest the existence of human prion strains. For the search of medical compounds an adequate assay, that is based on the human pathogenesis, suitable for high-throughput screening and highly reproducible, is missing. The real-time quaking-induced conversion (RT-QuIC), a recently developed in-vitro-method, allows the proof of, so far not-detectable, amounts of PrPSc in human cerebrospinal fluid (CSF). For this purpose self-propagating properties of PrPSc get used. First examinations suggest distinct properties of human prion diseases in RT-QuIC. To get used in diagnostic and research RT-QuIC needs to get evaluated and optimized for applications with CSF. Sensitivity of RT-QuIC is 85.5 % and specificity 99.5 % for the analysis of CSF. Reproducibility shows an almost perfect agreement. Pre-analytical short-term storage of CSF at room temperature or +4°C and long-term storage at −80°C as well as repeated freezing and thawing cycles do not influence the results. However contamination with blood leads to false-negative results. These findings suggest RT-QuIC as a suitable tool for prion diagnosis in the lifetime of the patient. To characterize the influence of putative prion disease strains, CSF-samples of different human prion diseases, e. g. sporadic and genetic forms, were analyzed by RT-QuIC. The results show distinct properties of PrPSc in CSF, modulated by the type of prion disease, PRNP codon 129 genotype and disease duration. These findings show the potential of RT-QuIC to examine the self-propagating properties of PrPSc in CSF, providing for the first time a method to study these effects in patients during the symptomatic phase. To examine the potential of RT-QuIC to get used as a drug screening test, the influence of several substances on the RT-QuIC response has been investigated. Doxycycline inhibits the reaction as well in correlation to the dose as in correlation to the time of add-on. These findings suggest the suitability of RT-QuIC to search for substances, which inhibit the conversion of PrP and show the inhibiting effect of doxycycline on the in-vitro-amplification of PrPSc.