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.