Release of Full-Length PrP(C) from Cultured Neurons Following Neurotoxic Challenges.

Kevin K W Wang,Zhiqun Zhang,Richard Rubenstein,Allen Chiu,J Susie Zoltewicz

doi:10.3389/fneur.2012.00147

Kevin K W Wang, Zhiqun Zhang + Show 3 more

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https://doi.org/10.3389/fneur.2012.00147

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Abstract

The susceptibility of the normal cellular prion protein isoform, cellular prion protein (PrPC), to proteolytic digestion has been well documented. In addition, a link between PrPC and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP) was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrPC-expressing mice were treated with calpain, no appreciable loss of the intact PrPC, nor the appearance of PrPC breakdown products (BDPs) were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC) were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX) and N-methyl-d-aspartate (NMDA), PrPC-BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrPC is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrPC. Similarly, the lysosomal protease inhibitor, NH4Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrPC or its release from the RtCNC. In conclusion, rat neuronal PrPC is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrPC into the cell culture media suggests an unidentified neuroprotective mechanism for PrPC.

Highlights

The cellular prion protein (PrPC) is a host-coded, glycosylphosphatidylinositol (GPI)-anchoredtransmembrane glycoprotein found in all mammalian cells with relatively high levels in the central nervous system (CNS; Kretzschmar et al, 1986; Moudjou et al, 2001) at neuronal synapses (Sales et al, 1998)
Following SDS-PAGE and Coomassie blue staining, intact (His)6-human recombinant PrP (HrPrP) migrated as an ∼30 kDa protein, while four major membrane and stained with Coomassie blue. (A) Major breakdown products (BDPs) were identified by N-terminal microsequencing. (B) Based on the PrPC amino acid sequence the cleavage sites are identified and depicted by downward arrows
We performed in vitro and in cell-based studies using primary rat cerebrocortical neuronal cultures (RtCNC) to further examine this issue. (His)6-HrPrP was cleaved by calpain-1 and -2 in vitro and the two targeted cleavage sites were identified between resides Gly40-Ser41 and residues Gly130-Ser131. (His)6-HrPrP, was resistant to caspase-3 digestion

Summary

Introduction

The cellular prion protein (PrPC) is a host-coded, glycosylphosphatidylinositol (GPI)-anchored (linked through Serine-231)transmembrane glycoprotein found in all mammalian cells with relatively high levels in the central nervous system (CNS; Kretzschmar et al, 1986; Moudjou et al, 2001) at neuronal synapses (Sales et al, 1998). The protease-sensitive PrPC is converted into the conformationally altered, disease-specific PrPSc isoform which is partially proteaseresistant (Riesner, 2003). PrPC has been associated with numerous cellular roles associated with cell signaling and/or neuroprotective functions, the specific physiologic function of PrPC remains unclear. PrPC neuroprotective functions that have been demonstrated both in vivo and in vitro (Westergard et al, 2007; Linden et al, 2008). PrPC protects neurons in vitro and in vivo from N -methyl-d-aspartate (NMDA)-evoked excitotoxicity (Khosravani et al, 2008) and against oxidative stress and plays a role in Cu/Zn superoxide dismutase activity (Brown and Besinger, 1998; Wong et al, 2000; Brown et al, 2002)

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