Calpain-dependent Endoproteolytic Cleavage of PrPSc Modulates Scrapie Prion Propagation

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 scrapieinfected 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.

Calpain Inhibition Is Sufficient to Suppress Aggregation of Polyglutamine-expanded Ataxin-3

“…DNA was routinely prepared from Escherichia coli SURE cells (Stratagene). The pRK5 expression plasmid with human full-length calpastatin cDNA was a kind gift from Dr. Glenn C. Telling, University of Kentucky, Lexington (30).…”

The P’s and Q’s of cellular PrP-Aβ interactions

“…33,34 C2-PrP is seen readily in prion infections and less abundant in normal brain whereas C1 is readily detected in normal brain samples. 35,36 The concept of a "switch" or decision point hinging upon processing of PrP to N1/C1 vs. to N2/C2 (and hence the relative stoichiometries of these fragments) may also have validity when one considers cellular insults other than prion infections. For example, upon exposure to staurosporine or an ischemia paradigm the N1 fragment of PrP is noted as being protective 37 while C1-PrP, but not C2-PrP, has a pro-apoptotic effect.…”

Cell models of prion infection

“…Little is known on the cellular proteases involved in processing and/or degradation of abnormal PrP. However, some protease inhibitors can modulate the processing [152] or the degradation [79] of PrP Sc in infected cultures and the tyrosine kinase inhibitor STI571 promotes lysosomal degradation of PrP Sc [42]. A delicate balance between synthesis and degradation of PrP Sc could be a determining factor for successful multiplication in cultured cells [150].…”

“…Monoclonal antibody 6H4 (recognising residues [144][145][146][147][148][149][150][151][152] and Fab fragments of D18 and D13 mAbs (recognising residues 132-156 and 95-103, respectively) potently inhibit prion multiplication in N2a cells [41,102]. SAF34 and SAF61 mAbs, which recognise the octarepeat region and residues 114-152 respectively, impaired prion propagation in infected neuroblastoma cells with an increase of the turn-over of PrP C as a proposed mechanism for prion inhibition [104].…”