Abstract
Previous studies using post-mortem human brain extracts demonstrated that PrP in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27-30), the protease-resistant core of PrP(Sc) (1). The role of this endoproteolytic cleavage of PrP in prion pathogenesis and the identity of the cellular protease responsible for production of the C2 cleavage product has not been explored. To address these issues we have taken a combination of pharmacological and genetic approaches using persistently infected scrapie mouse brain (SMB) cells. We confirm that production of C2 is the predominant cleavage event of PrP(Sc) in the brains of scrapie-infected mice and that SMB cells faithfully recapitulate the diverse intracellular proteolytic processing events of PrP(Sc) and PrP(C) observed in vivo. While increases in intracellular calcium (Ca(2+)) levels in prion-infected cell cultures stimulate the production of the PrP(Sc) cleavage product, pharmacological inhibitors of calpains and overexpression of the endogenous calpain inhibitor, calpastatin, prevent the production of C2. In contrast, inhibitors of lysosomal proteases, caspases, and the proteasome have no effect on C2 production in SMB cells. Calpain inhibition also prevents the accumulation of PrP(Sc) in SMB and persistently infected ScN2A cells, whereas bioassay of inhibitor-treated cell cultures demonstrates that calpain inhibition results in reduced prion titers compared with control-treated cultures assessed in parallel. Our observations suggest that calpain-mediated endoproteolytic cleavage of PrP(Sc) may be an important event in prion propagation.
Highlights
Previous studies using post-mortem human brain extracts demonstrated that prion protein (PrP) in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27–30), the protease-resistant core of PrPSc [1]
We used a combination of pharmacological and genetic approaches to ascertain the nature of the cellular protease responsible for PrPSc cleavage and to address the role of the C2 cleavage product in the conversion of PrPC to PrPSc and prion pathogenesis
While pharmacological inhibitors of calpains prevented the production of C2, inhibitors of lysosomal proteases, caspases and the proteasome had no effect on C2 production in scrapie mouse brain (SMB) cells
Summary
Chemicals and Antibodies—All immunoblots probed with Fab D-18 were developed using horseradish peroxidase-conjugated goat anti-Hu secondary antibody and ECL or ECL-Plus detection (Amersham Biosciences) and exposed to x-ray film. For Ca2ϩ ionophore treatments, the culture medium of subconfluent monolayers of SMB cells was replaced for 1 h with Optimem (Invitrogen) containing 2 mM CaCl2 and various concentrations of ionomycin or A23187, after which detergent extracts were prepared. For PrP-(27–30) analysis in brain extracts, homogenates containing 50 g of total protein were treated with PK. When cells achieved confluence they were trypsinized and reseeded onto 6-well plates, and the following day inhibitor-containing medium was replaced with normal medium containing 1 g/ml calcein-AM and propidium iodide dyes for 30 min at 37 °C. For inoculation of Swiss CD-1 mice, 10% (w/v) homogenates of RML-infected mouse brain were prepared by repeated extrusion through an 18-gauge syringe needle followed by a 21-gauge needle in PBS lacking calcium and magnesium ions. All statistical analyses including the Student’s t tests were performed using GraphPad Prism version 4.0 for Windows, GraphPad Software, San Diego CA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
