Abstract
Transmissible spongiform encephalopathies, also known as prion diseases, are a group of fatal neurodegenerative disorders affecting both humans and animals. The central pathogenic event in prion disease is the misfolding of normal prion protein (PrPC) into the pathogenic conformer, PrPSc, which self-replicates by converting PrPC to more of itself. The biochemical hallmark of PrPSc is its C-terminal resistance to proteinase K (PK) digestion, which has been historically used to define PrPSc and is still the most widely used characteristic for prion detection. We used PK-resistance as a biochemical measure for the generation of recombinant prion from bacterially expressed recombinant PrP. However, the existence of both PK- resistant and -sensitive PrPSc forms in animal and human prion disease led to the question of whether the in vitro-generated recombinant prion infectivity is due to the PK-resistant or -sensitive recombinant PrP forms. In this study, we compared undigested and PK-digested recombinant prions for their infectivity using both the classical rodent bioassay and the cell-based prion infectivity assay. Similar levels of infectivity were detected in PK-digested and -undigested samples by both assays. A time course study of recombinant prion propagation showed that the increased capability to seed the conversion of endogenous PrP in cultured cells coincided with an increase of the PK-resistant form of recombinant PrP. Moreover, prion infectivity diminished when recombinant prion was subjected to an extremely harsh PK digestion. These results demonstrated that the infectivity of recombinant prion is encoded within the structure of the PK-resistant PrP fragments. This characteristic of recombinant prion, that a simple PK digestion is able to eliminate all PK-sensitive (non-infectious) PrP species, makes possible a more homogenous material that will be ideal for dissecting the molecular basis of prion infectivity.
Highlights
Prion diseases are a group of fatal neurodegenerative disorders affecting both human and animals [20]
Amplification of recPrPSc in vitro was accompanied by an increase of prion infectivity Previously, we demonstrated that recPrPSc generated through serial protein misfolding cyclic amplification (sPMCA) with recPrP and two cofactors— 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′rac-glycerol) (POPG) and total RNA isolated from normal mouse liver—is highly infectious to wild-type mice and has an infectious dose 50 (ID50) around 104/μg recPrP [36, 38]
It has been used to reveal the relationship between prion infectivity and neurotoxicity [27] and the evolution of a prion when it is exposed to changing environments [11]
Summary
Prion diseases are a group of fatal neurodegenerative disorders affecting both human and animals [20]. Prions were once considered an unorthodox diseasecausing agent, but it is clear that in other common, late-onset, neurodegenerative diseases, protein aggregates such as Aβ and tau in Alzheimer’s disease and αsynuclein in Parkinson’s disease are able to propagate their diseased conformations in a “prion-like” manner [8, 21, 31]. Among all these protein aggregates, PrPSc is the only one that has been proven to be a diseasecausing agent. Elucidating the molecular mechanism of prion infectivity is important for combatting prion disease, but will shed light on the molecular mechanisms of propagation of other protein aggregates
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