Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrP^Sc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrP^Sc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrP^Sc). Although evidence suggests that sPrP^Sc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte''s ability to degrade PrP^Sc. Furthermore, the cell does not appear to distinguish between sPrP^Sc and protease-resistant PrP^Sc, suggesting that sPrP^Sc could contribute to prion infection.Prion diseases, or transmissible spongiform encephalopathies, are rare fatal neurologic disorders of mammals that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep. Prion diseases are characterized by the conversion of normal prion protein (PrP^C) into a disease-associated and aggregated isoform (PrP^Sc), which is thought to be the main component of the infectious agent or prion (reviewed in Priola and Vorberg¹). PrP^C is a glycoprotein that contains two N-linked glycosylation sites^2,3 and is bound to the plasma membrane via a glycosyl-phosphatidyl-inositol anchor.⁴ Although PrP^C is detergent soluble and fully susceptible to proteolytic degradation, PrP^Sc has an increased detergent insolubility and partial resistance to proteinase K (PK).⁵ The presence of amino-terminally truncated, PK-resistant core fragments of PrP^Sc (rPrP^Sc) after limited proteolysis is considered the most reliable diagnostic marker for prion infection,^6,7 and biochemical profiles of rPrP^Sc based on molecular mass and/or the degree of glycosylation are used to help differentiate distinct prion disease phenotypes in humans.^8–11In recent years, alternative approaches for analyzing PrP^Sc that do not rely on the enzymatic removal of PrP^C have indicated that not all forms of PrP^Sc are necessarily resistant to proteolytic treatment. The conformation-dependent immunoassay, which uses conformational differences between the N termini of PrP^C and PrP^Sc, has found that often a majority of PrP^Sc present in prion-affected brains is susceptible to proteolytic degradation.^12–15 In sporadic CJD (sCJD), this PK-sensitive species of aggregated PrP^Sc, termed sPrP^Sc, was found in some cases to account for up to 90% of the total PrP^Sc.^13,16 Careful analysis of the size distribution of PrP^Sc has also found that sPrP^Sc forms much smaller aggregates than rPrP^Sc.^17,18 Thus, sPrP^Sc appears to represent a population of PrP^Sc aggregates which tends to be both smaller and more protease sensitive than rPrP^Sc.Evidence suggests that, like rPrP^Sc, sPrP^Sc has seeding activity and can convert PrP^C to protease resistance.^18,19 It has also been associated with prion infectivity²⁰ and may influence the incubation time of prion disease.²¹ Although these data suggest that PK-sensitive disease-associated PrP aggregates may be actively involved in prion pathogenesis, no studies have been performed to determine how this population of PrP aggregates might interact with cells and influence prion infection. In this study, we have looked at the uptake and degradation of PK-sensitive and PK-resistant disease-associated PrP aggregates in an established human astrocyte cell line. Our results indicate that, despite their biochemical differences, PK-sensitive PrP aggregates are taken up and degraded similarly to PK-resistant PrP aggregates, suggesting that relative PK resistance does not significantly influence the cell''s ability to degrade PrP^Sc. Thus, the astrocyte does not appear to distinguish between sPrP^Sc and rPrP^Sc aggregates, suggesting that sPrP^Sc could be involved in prion pathogenesis.