We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.
The accumulation of PrPSc in scrapie‐infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA‐expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti‐LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrPSc in these cells. The treatments also reduced PrPc levels. The anti‐LRP/LR antibody, W3, abolished PrPSc accumulation and reduced PrPc levels after seven days of incubation. Cells remained free of PrPSc after being cultured for 14 additional days without the antibody, whereas the PrPc level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrPSc propagation in cultured cells and suggest that LRP/LR‐specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.
Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virushost interactions. Recently, we demonstrated that the IFN-induced 3 0 -5 0 exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-kB factors p50 and p65 bind and activate the kB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-kB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.
The 37/67 kDa laminin receptor (LRP/LR) acts as a receptor for prions providing a promising target for the treatment of prion diseases. Recently, we selected anti-LRP/LR single-chain antibodies (scFvs) and proved a reduction of the peripheral PrP Sc propagation by passive immunotransfer into scrapie-infected mice. Here, we report the development of an in vivo gene delivery system based on adeno-associated virus (AAV) vectors expressing scFvs-S18 and -N3 directed against LRP/LR. Transduction of neuronal and non-neuronal cells with recombinant (r)AAV serotype 2 vectors encoding scFv-S18, -N3 and -C9 verified the efficient secretion of the antibodies. These vectors were administered via stereotactic intracerebral microinjection into the hippocampus of C57BL/6 mice, followed by intracerebral inoculation with 10 % RML at the same site 2 weeks post-injection of rAAV. After 90 days post-infection, scFv-S18 and -N3 expression resulted in the reduction of peripheral PrP Sc propagation by approximately 60 and 32 %, respectively, without a significant prolongation of incubation times and survival. Proof of rAAV vector DNA in spleen samples by real-time PCR strongly suggests a transport or trafficking of rAAV from the brain to the spleen, resulting in rAAV-mediated expression of scFv followed by reduced PrP Sc levels in the spleen most likely due to the blockage of the prion receptor LRP/LR by scFv.Prion diseases are fatal lethal neurodegenerative diseases affecting humans and animals (for review see Weissmann, 2004;Zuber et al., 2007a). None of the affected individuals can be treated or cured effectively (Ludewigs et al., 2007;Vana et al., 2007;Weissmann & Aguzzi, 2005). The abnormal form of the prion protein, PrP Sc , is frequently associated with infectivity and propagates mainly in the brain and the lymphoreticular system (LRS). Accumulation of the aggregated PrP Sc leads to neuronal death. PrP Sc is distinct from the host protein PrP c by its biochemical properties such as proteinase K sensitivity and insolubility, but harbours the same amino acid sequence. The generation of PrP Sc from PrP c involves conformational changes accompanied by modifications in the secondary structure of the protein (for review see Aguzzi & Weissmann, 1998;Prusiner, 1998;Weissmann, 2004).The 37/67 kDa laminin receptor (LRP/LR) is a multifunctional protein (i) playing an important role in cell adhesion, movement and growth of many cell types, (ii) acting as a receptor for some subtypes of adeno-associated 3These authors contributed equally to this work. (Morel et al., 2005). The fact that LRP levels are increased in organs of the LRS and central nervous system (CNS), such as spleen and brain, of infected animals (Rieger et al., 1997) strongly suggests that this receptor is not only essential for prion uptake after oral infection but also plays an important role for PrP Sc propagation and prion pathogenesis in the peripheral nervous system, including the LRS and CNS. Additionally, several laminin receptor isoforms have been found in mouse bra...
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