Prion interaction with the 37-kDa/67-kDa laminin receptor on enterocytes as a cellular model for intestinal uptake of prions.

Enterocytes, a major cell population of the intestinal epithelium, represent one possible barrier to the entry of prions after oral exposure. We established a cell culture system employing enterocytes from different species to study alimentary prion interaction with the 37-kDa/67-kDa laminin receptor LRP/LR. Human, bovine, porcine, ovine, and cervid enterocytes were cocultured with brain homogenates from cervid, sheep, and cattle suffering from chronic wasting disease (CWD), scrapie, and bovine spongiform encephalopathy (BSE), respectively. PrP(CWD), ovine PrP(Sc), and PrP(BSE) all colocalized with LRP/LR on human enterocytes. PrP(CWD) failed to colocalize with LRP/LR on bovine, porcine, and ovine enterocytes. Ovine PrP(Sc) colocalized with the receptor on bovine enterocytes, but failed to colocalize with LRP/LR on cervid and porcine enterocytes. PrP(BSE) failed to colocalize with the receptor on cervid and ovine enterocytes. These data suggest possible oral transmissibility of CWD and sheep scrapie to humans and may confirm the oral transmissibility of BSE to humans, resulting in zoonotic variant Creutzfeldt-Jakob disease. CWD might not be transmissible to cattle, pigs, and sheep. Sheep scrapie might have caused BSE, but may not cause transmissible spongiform encephalopathy in cervids and pigs. BSE may not be transmissible to cervids. Our data recommend the enterocyte model system for further investigations of the intestinal pathophysiology of alimentary prion infections.

Scrapie-infected transgenic mice expressing a laminin receptor decoy mutant reveal a prolonged incubation time associated with low levels of PrPres.

The 37-kDa/67-kDa laminin receptor (LRP/LR) was identified as a cell surface receptor for prion proteins. The laminin receptor mutant LRP102-295::FLAG interfered with PrP(Sc) propagation in murine neuronal cells presumably acting as a decoy in a transdominant negative fashion by trapping PrP molecules in the extracellular matrix. Here, we generated hemizygous transgenic mice expressing LRP102-295::FLAG in the brain. Scrapie-infected transgenic mice exhibit a significantly prolonged incubation time in comparison to scrapie-infected wild-type (FVB) mice. At the terminal stage, transgenic mice revealed significantly reduced proteinase-K-resistant PrP levels by 71% compared to wild-type mice. Our results recommend the laminin receptor decoy mutant as an alternative therapeutic tool for treatment of transmissible spongiform encephalopathies.

LRP/LR as an alternative promising target in therapy of prion diseases, Alzheimer's disease and cancer.

The 37 kDa/67 kDa laminin receptor (LRP/LR) represents a key player for cell adhesion, is associated with the metastatic potential of solid tumors and is required for maintenance of cell viability by preventing apoptosis. LRP/LR acts as a receptor for viruses such as Sindbis virus, Venezuelean Equine Encephalitis (VEE) virus, Adeno-associated-viruses (AAV) and Dengue Virus, the latter causing 50 to 100 million infections in humans per year. LRP/LR acts further as a receptor for prions and represents a multifunctional protein subcellularly located to the nucleus, the cytoplasm and the cell surface. The receptor represents an alternative target for therapy of viral infections, cancer and prion disorders and might play additional roles in further neurodegenerative diseases such as Alzheimer's disease. The species barrier in prion disorders might be at least in part determined by the presence of LRP/LR in enterocytes of the intestinal epithelium. Anti-LRP/LR antibodies, siRNAs directed against LRP mRNA, polysulfated glycanes such as pentosan polysulfate and heparan mimetics and LRP decoy mutants are promising tools for blocking or downregulating the receptor and may represent alternative therapeutics for the treatment of prion disorders, Alzheimer's Disease and metastatic cancer.

Microinjection of lentiviral vectors expressing small interfering RNAs directed against laminin receptor precursor mRNA prolongs the pre-clinical phase in scrapie-infected mice.

We examined therapeutic in vitro and in vivo approaches using lentivirus-based packaging of small interfering RNAs (siRNAs) targeting the non-integrin laminin receptor mRNA for treatment and prevention of prion disorders. Transfection of N2aSc(+) cells with recombinant plasmids expressing three different siRNAs, significantly reduced both the LRP (laminin receptor precursor) and PrP(Sc) levels by approximately 40-60 %. Stereotactic intracerebral microinjection of recombinant lentiviral vectors LVsiRNA-LRP 7 and 9 into the cortex of C57BL/6 wild-type mice resulted in a significant reduction of the LR levels in the cortex 15 days post-injection by 62 and 82 %, respectively. Intracerebral RML inoculation of C57BL/6 mice after microinjection with recombinant lentiviral vector LVsiRNA-LRP 7 into the hippocampus resulted in a significant reduction of both LRP and PrP(Sc) levels by 36 and 41 %, respectively, concomitant with a significant prolongation of the pre-clinical phase. Lentiviral vectors expressing siRNAs targeting LRP mRNA represent a novel delivery system for the treatment of transmissible spongiform encephalopathies.

Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins.

The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-DeltaSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-DeltaSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules.

Therapeutic approaches for prion disorders.

Prion diseases are lethal for both humans and animals, and affected individuals die after several months following a rapid disease progression. Although researchers have attempted for decades to develop effective therapeutics for the therapy of human prion disorders, until now no efficient drug has been available on the market for transmissible spongiform encephalopathy (TSE) treatment or cure. Approximately 200 patients worldwide have died or suffer from variant Creutzfeldt-Jakob disease (CJD). Incidences for sporadic and familial CJD are approximately 1.5-2 per million per year and one per 10 million per year, respectively, in Europe. This review summarizes classical and modern trials for the development of effective anti-TSE drugs, introduces potential effective delivery systems, such as lentiviral and adeno-associated virus systems for antiprion components, including antibodies and siRNAs, and presents vaccination trials. Most of the antiprion drugs target prion protein PrP(c) and/or PrP(Sc). Alternative targets are receptors and coreceptors for PrP, that is, the 37/67-kDa laminin receptor and heparan sulfate proteoglycanes. We review clinical trials for the treatment of TSEs and describe hindrances and chances for a breakthrough in therapy of prion disorders.

TNF-dependent overexpression of CCL21 is an underlying cause of progressive lymphoaccumulation in generalized lymphoproliferative disorder.

The human condition autoimmune lymphoproliferative syndrome and the murine mutation generalized lymphoproliferative disorder (gld/gld) are both caused by mutations of Fas or Fas ligand and are characterized by severe splenomegaly and lymphadenopathy. In the mouse, the additional absence of TNF attenuates the gld/gld syndrome through an unknown mechanism. We hypothesized that this unexpected outcome was not mediated by increased apoptosis but changes of T cell localization. We demonstrated that the homeostatic chemokine CCL21 is strongly up-regulated in the spleen of C57BL/6 (B6).gld/gld and B6.gld/gld.TRAIL-/- mice. In contrast, a distinct consequence of TNF deficiency in B6.gld/gld mice was the substantially reduced splenic production of CCL21. An analysis of the cognate chemokine receptor CCR7 showed a complete, age-dependent down-regulation of this receptor on B6.gld/gld conventional peripheral T cells that are therefore unable to react to this chemokine. These results demonstrate a new role for the pro-inflammatory cytokine TNF and the TNF-regulated chemokine CCL21 in the complex etiology of the autoimmune syndrome in B6.gld/gld mice.

Novel aspects of prions, their receptor molecules, and innovative approaches for TSE therapy.

1. Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible spongiform encephalopathies (TSEs), that affect both animals and humans and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. TSEs are usually rapidly progressive and clinical symptoms comprise dementia and loss of movement coordination due to the accumulation of an abnormal isoform (PrP(Sc)) of the host-encoded prion protein (PrP(c)). 2. This article reviews the current knowledge on PrP(c) and PrP(Sc), prion replication mechanisms, interaction partners of prions, and their cell surface receptors. Several strategies, summarized in this article, have been investigated for an effective antiprion treatment including development of a vaccination therapy and screening for potent chemical compounds. Currently, no effective treatment for prion diseases is available. 3. The identification of the 37 kDa/67 kDa laminin receptor (LRP/LR) and heparan sulfate as cell surface receptors for prions, however, opens new avenues for the development of alternative TSE therapies.

A trans-dominant negative 37kDa/67kDa laminin receptor mutant impairs PrP(Sc) propagation in scrapie-infected neuronal cells.

The 37kDa/67kDa laminin receptor (LRP/LR) has been identified as a cell surface receptor for cellular and infectious prion proteins. Here, we show that an N-terminally truncated LRP mutant encompassing the extracellular domain of the LRP/LR (LRP102-295::FLAG) reduces the binding of recombinant cellular huPrP to mouse neuroblastoma cells, and infectious moPrP27-30 to BHK cells, and interferes with the PrP(Sc) propagation in scrapie-infected neuroblastoma cells (N2aSc(+)). A cell-free binding assay demonstrated the direct binding of the LRP102-295::FLAG mutant to both PrP(c) and PrP(Sc). These results, together with the finding that endogenous LRP levels remain unaffected by the expression of the mutant, indicate that the secreted LRP102-295::FLAG mutant may act in a trans-dominant negative manner as a decoy by trapping PrP molecules. The LRP mutant might represent a potential therapeutic tool for the treatment of TSEs.

Knock-down of the 37-kDa/67-kDa laminin receptor in mouse brain by transgenic expression of specific antisense LRP RNA.

The 37-kDa/67-kDa laminin receptor (LRP/LR) plays a major role in the propagation of PrPSc, the abnormal form of the prion protein. In order to ablate the expression of LRP/LR in mouse brain we generated transgenic mice ectopically expressing antisense LRP RNA in the brain under control of the neuron-specific enolase (NSE) promoter. Hemizygous transgenic mice TgN(NSEasLRP)2 showed a significant reduction of LRP/LR protein levels in hippocampal and cerebellar brain regions. These mice might act as powerful tools to investigate the role of the laminin receptor in scrapie pathogenesis.

The 37 kDa/67 kDa laminin receptor is required for PrP(Sc) propagation in scrapie-infected neuronal cells.

The accumulation of PrP(Sc) 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 PrP(Sc) in these cells. The treatments also reduced PrP(c) levels. The anti-LRP/LR antibody, W3, abolished PrP(Sc) accumulation and reduced PrP(c) levels after seven days of incubation. Cells remained free of PrP(Sc) after being cultured for 14 additional days without the antibody, whereas the PrP(c) level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP(Sc) 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.