Abstract
Sporadic Creutzfeldt-Jakob disease (sCJD) is a transmissible, rapidly progressive and fatal neurodegenerative disease. The transmissible agent linked to sCJD is composed of the misfolded form of the host-encoded prion protein. The combination of histopathological and biochemical analyses has allowed the identification and sub-classification of six sCJD subtypes. This classification depends on the polymorphic variability of codon 129 of the prion protein gene and the PrPres isotype, and appears to be associated with neuropathological and clinical features. Currently, sCJD subtyping is only fully achievable post mortem. However, a rapid and non-invasive method for discriminating sCJD subtypes in vita would be invaluable for the clinical management of affected individuals, and for the selection of participants for clinical trials. The CSF analysis by Real Time Quaking Induced Conversion (RT-QuIC) reaction is the most sensitive and specific ante mortem sCJD diagnostic test available to date, and it is used by a number of laboratories internationally. RT-QuIC takes advantage of the natural replication mechanisms of prions by template-induced misfolding, employing recombinant prion protein as reaction substrate. We asked whether epitope mapping, of the RT-QuIC reaction products obtained from seeding RT-QuIC with brain and CSF samples from each of the six molecular subtypes of sCJD could be employed to distinguish them and therefore achieve in vita sCJD molecular subtyping. We found that it is possible to distinguish the RT-QuIC products generated by sCJD biological samples from the ones generated by spontaneous conversion in the negative controls, but that different sCJD subtypes generate very similar, if not identical RT-QuIC reaction products. We concluded that whilst RT-QuIC has demonstrable diagnostic value it has limited prognostic value at this point in time.
Highlights
Prions are the infectious agents associated with Transmissible Spongiform Encephalopathies (TSEs) or prion diseases
These findings suggest that Sporadic Creutzfeldt-Jakob disease (sCJD) subtypes may be discriminated by Real Time Quaking Induced Conversion (RT-QuIC) based on the biochemical characteristics of the generated aggregates, it is not clear at this point in time whether the commonly employed routine cerebrospinal fluid (CSF) analysis by RT-QuIC can be extended to subtype sCJD using the same criteria used for post-mortem PrPres subtyping from brain tissue
We initially generated RT-QuIC reaction products using sCJD 10% brain homogenates (10%BH) as RT-QuIC seeds, and analysed them for their resistance to protease K (PK) digestion
Summary
Prions are the infectious agents associated with Transmissible Spongiform Encephalopathies (TSEs) or prion diseases. A recent adaptation of RT-QuIC, employing an N-terminally truncated form of the Hamster recombinant PrP (i.e. 90-231Ha-rPrP) as a reaction substrate, revealed sCJD subtype-specific kinetics of aggregation, when reactions were seeded with CSF samples [26]. There is evidence that the misfolded form(s) of recombinant PrP generated during the RT-QuIC reaction ( referred to as RT-QuIC reaction products) can retain structural features of the prion seed that generated them allowing for their in vitro identification [27,28,29] These findings suggest that sCJD subtypes may be discriminated by RT-QuIC based on the biochemical characteristics of the generated aggregates, it is not clear at this point in time whether the commonly employed routine CSF analysis by RT-QuIC can be extended to subtype sCJD using the same criteria used for post-mortem PrPres subtyping from brain tissue. By seeding the diagnostic RT-QuIC reaction with brain and CSF samples accounting for the most common sCJD subtypes (MM1/MV1, MM2, MV2, VV1, VV2), we have analysed the RT-QuIC reaction products for their resistance to limited proteolysis and investigated the length of the resistant fragments via epitope mapping using a set of six commercially available antibodies, including those usually employed for sCJD molecular typing
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