Proteoglycans of reactive rat cortical astrocyte cultures: abundance of N-unsubstituted glucosamine-enriched heparan sulfate.

"Reactive" astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH2 to GalNH2, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a >5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~20% and ~40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.

GFP-tagged PrP supports compromised prion replication in transgenic mice.

The ability of green fluorescent protein (GFP)-prion protein (PrP) fusions to support prion propagation has not been demonstrated. Here, we show that while transgenic mice expressing PrP tagged at its amino terminus with enhanced GFP, referred to as EGFPrP-N, supported prion replication, disease onset was prolonged, the brains of diseased mice did not exhibit typical disease neuropathology and disease-associated EGFPrP-N lacked the full spectrum of biochemical properties normally associated with PrP(Sc). Co-expression of wild-type PrP and EGFPrP-N substantially reduced prion incubation times and resulted in accumulation of protease-resistant EGFPrP(Sc)-N in the brains of transgenic mice as well as chronically infected cultured cells, suggesting that wild-type PrP rescued a compromised amino terminal function in EGFPrP-N. While our results show that EGFPrP(C)-N adopts a conformation necessary for the production of infectious prions, the synergistic interaction of wild-type and EGFPrP-N underscores the importance of the amino terminus in modulating prion pathogenesis.

Calpain-dependent endoproteolytic cleavage of PrPSc modulates scrapie prion propagation.

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.