Podcasts about lrp lr

Fri, 15 May 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13091/ https://edoc.ub.uni-muenchen.de/13091/1/Pflanz_Heike.pdf Pflanz, Heike ddc:540, ddc:500, Fakultät für

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 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. A common hallmark of TSEs is the accumulation of an abnormal isoform (PrPSc) of the host-encoded prion protein (PrPc) in the brains of affected animals and humans. PrPc is a highly conserved cell surface sialoglycoprotein that is expressed in several cell types, mainly neuronal cells, but its normal physiological function is still not known. However, PrPc is elementary for the acquisition and the replication of prion diseases. Several inhibitors of the PrPSc formation have been reported, but none of them showed great potency in an in vivo application. Thus, the identification of the 37kDa/67kDa laminin receptor (LRP/LR) as the cell surface receptor for prions opened a new direction for the development of a TSE therapy. Currently, no treatment to slow down or stop the disease process in humans with any form of CJD is established. However, several strategies have been investigated to find an anti-prion treatment including development of a vaccination therapy and screening for potent chemical compounds. In scrapie-infected neuronal cells, which represent a widely used and well characterized in vitro model for transmissible spongiform encephalopathies, the accumulation of PrPSc has been prevented by transfection of (i) antisense LRP RNA, (ii) small interfering RNAs targeting the LRP mRNA and (iii) incubation with the polyclonal anti-LRP antibody W3. Furthermore, the knock down of surface LRP/LR resulted in a reduction of the cellular PrP levels, suggesting an interference with the PrP internalization process. Thus, LRP/LR is required for the PrPSc propagation in vitro and involved in the PrPc metabolism.Due to the existence of several LR genes, a major step to investigate the role of the 37kDa/67kDa laminin receptor in scrapie pathogenesis in vivo is the generation of transgenic mice exhibiting a lower level of LRP/LR. Hemizygous transgenic mice that express LRP/LR antisense RNA under the control of the neuron-specific enolase (NSE) promoter were generated and showed a reduced LRP/LR protein level in the cerebellum and the hippocampus. Intracerebral inoculation of these transgenic mice with the scrapie agent will show, whether the accumulation of pathogenic PrPSc in the brain is delayed or prevented due to a reduced LRP/LR level. A further therapeutic anti-prion approach is given by LRP/LR deletion mutants that can be secreted to the cell culture medium and might act as decoys. Previously, it has been demonstrated that a transmembrane deletion mutant is able to prevent PrPc binding and internalization. In vitro studies using an N-terminally truncated LRP mutant, representing the extracellular domain of LRP/LR (LRP102-295::FLAG), revealed a reduced binding of (i) recombinant cellular PrP to mouse neuroblastoma cells, (ii) infectious moPrP 27-30 to BHK21 cells and (iii) interfered with the PrPSc propagation in chronically scrapie-infected mouse neuroblastoma cells. Furthermore, a cell free binding assay demonstrated the direct binding of the LRP102-295::FLAG mutant to both PrPc and PrPSc. These results together with the finding that 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. To investigate the therapeutic potential of the LRP102-295::FLAG decoy mutant in vivo transgenic mice were generated ectopically expressing LRP102-295::FLAG in the brain. Animals showed no phenotype and transgene expression was detected in cortical and cerebellar brain regions. An intracerebral prion inoculation of these mice will prove whether the expression of the LRP102-295::FLAG mutant can impair the PrPSc accumulation in the brain and can thus, act as a alternative therapeutic tool in prion diseases. The recent finding that experimental introduction of RNA can be used to interfere with the function of an endogenous gene (RNA interference) provided another tool for the development of gene-specific therapeutics. In order to evaluate a gene transfer therapeutic TSE strategy, human immunodeficiency virus (HIV)-derived vectors that express short hairpin RNA (shRNA) directed against the LRP mRNA were used. Following integration of LRP-shRNA-expressing lentiviral vectors into the genome of neuronal cells efficient LRP/LR downregulation was observed. In scrapie infected neuronal cells, downregulation of the LRP gene expression resulted in a diminishment of PrPSc propagation, providing a further therapeutic strategy in the development of a TSE treatment.

Prions are unconventional pathogens that cause transmissible spongiform encephalopathies (TSEs). According to the "protein only" hypothesis, prions consist of an infectious protein that is capable of converting a normal host protein termed PrPc into a protease resistant form termed PrPSc. PrPSc is poorly degraded by the host and accumulates in the CNS. Normal biological functions of PrPc and mechanisms involved in neurodegeneration remain obscure. During the past two decades, considerable efforts have been made to elucidate prion diseases and in particular to identify PrP interactors for a better understanding in prion biology. A major break-through was the identification of the 37 kDa laminin receptor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), as the cell surface receptor for the cellular prion protein. We investigated the role of LRP/LR in the propagation of PrPSc in chronically infected cells by different approaches. Three strategies resulted in downregulation or blocking of LRP and prevented PrPSc accumulation in different scrapie infected neuronal cell lines (i) transfection with an antisense LRP RNA expression plasmid (ii) transfection with small interfering (siRNAs) specific for the LRP mRNA and (iii) incubation with the polyclonal anti-LRP antibody, W3. We observed that the treatment with W3 abolished PrPSc deposition and reduced PrPc levels after one week of incubation. PrPSc did not reappear in cells being cultured for 14 additional days without therapeutic antibody treatment. Taken together, these results indicate that LRP is not only required for PrPc metabolism under non-pathological conditions but also has a pivotal role in prion propagation in a cell culture model. LRP/LR appears then to be a promising potential target for the development of therapeutics for the treatment of prion disease. Due to these encouraging cell culture data, we decided to select single chain antibodies (scFv) encompassing a suitable format for therapy. ScFvs are composed of variable parts of heavy and light chains of an immunoglobulin that are connected by a peptidic linker. The antibodies were screened on recombinant GST::LRP employing a phage display strategy. Two scFvs termed N3 and S18 were screened and selected by ELISA. Both antibodies were further characterized by western blotting and FACS analysis: both N3 and S18 specifically recognized mouseLRP and humanLRP overexpressed in mammalian cells under denaturating conditions (western blot) and under native conditions at the cell surface (FACS). Epitope mapping revealed that as expected both scFvs are directed against the extracellular part of LRP: S18 and N3 recognized amino acid residues 225-233 and 273-278, respectively. The ability of N3 and S18 to interfere with LRP/PrP interaction was tested by pull-down assays. In contrast to the control scFv C9 directed against the pre-S1 coat-protein of hepatitis B virus, both anti-LRP scFvs were able to block the specific LRP/PrP binding. In order to investigate a potential curing effect of scFv S18 in vivo, this scFv was tested in a scrapie mouse model by passive immunization. The application of S18 by intra-peritoneal injection was able to reduce PrPSc deposition in the spleen in comparison to mice injected with PBS or C9. However the survival times of S18 treated animals was not increased. Anti-LRP scFv S18 seems to contribute to block prion propagation in the periphery but it is likely that this effect was not enough strong to have an impact on the CNS invasion. Thus, we hypothesized that a strategy targeting directly the brain should be more effective. In this context, an approach based on the expression of single chain antibodies as secretory molecules in the brain via an adeno-associated virus (AAV) vector was initiated. To assure secretion of the scFv expressed in mammalian cells, a signal sequence was fused to the scFvs. Tranfection experiments demonstrated that neuronal cells were able to express and secrete high quantities of both scFvs. Furthermore, the generated scFvs were still functional as shown by western blotting. To find the appropriate AAV serotype for scFv expression, neuronal cells were transduced with varying serotypes carrying a GFP. AAV serotype 2 was chosen due to (i) its good transduction performance in two neuronal cell lines and (ii) the possibility of its purification by affinity chromatography. The sequences encoding for the scFvs N3, S18 and C9 have been cloned in an AAV-based vector. The AAV system was also able to drive high expression of scFvs into the supernatant by transfection or transduction. rAAV-scFv particles were produced and purifed for further stereotaxic injections into mice. Although the investigation of this therapeutic strategy is still in progress in a murine scrapie model, we already proved that a single injection of rAAV led to the expression of scFvs into the brain of mice 30 days post injection. This study represents the first gene therapeutic approach for the treatment of prion diseases.

Prionen Erkrankungen sind neurodegenerative Erkrankungen, bei denen die abnormale Form (PrPSc) des zellulären Prion Proteins (PrPC) eine entscheidente Rolle spielt. PrPSc lagert sich im Gehirn von Menschen und verschiedenen Säugetieren zu langen Ketten, sogenanntem Amyloid, zusammen und bildet amyloide Plaques. Dies führt letzendlich zum Tod des Individuums. Die Pathogenese und die zelluläre Funktion des Prion Proteins ist jedoch noch nicht vollständigig verstanden. Es wird vermutet, daß PrPC, sowie PrPSc mit verschieden Makromolekülen interagieren. In dieser Dissertation wird die Interaktion des 37-kDa/67-kDa Laminin-Rezeptor (LRP/LR) mit dem Prion Protein beleuchtet. Dabei konnte gezeigt werden, das beide Proteine an der Zelloberfläche von neuronalen Zellen interagieren und daß LRP/LR für die Internalisierung von rekombinantem PrPC notwending ist. Diese Internalisierung ist ein aktiver, rezeptorvermittelter Prozess. Darüberhinaus konnte gezeigt werden, daß LRP/LR notwendig für die Propagation des abnormalen Prion Proteins, PrPSc, in scrapie-infizierten neuronalen Zellen ist. Scrapie-infizierte neuronale Zellen stellen ein Modellsystem für PrPSc-Infektionen dar. Verschiedene Isoformen von LRP/LR aus Mäusehirn sind identifiziert worden und binden PrPC in overlay-assays. Damit konnte eine Interaktion aller Isoformen mit dem Prion Protein gezeigt werden. PrPC konnte in großen Mengen in der Hefe Pichia pastoris hergestellt werden. Dabei wurde sowohl eine monomere Form, als auch ein kovalent verknüpftes Dimer des Proteins von der Hefe produziert. Beide Proteine wurden biochemisch charakterisiert und stellen ein Substrat für Kristallisations- und Funktionsstudien dar.

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPSc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies (TSEs), which affect both, humans and animals. Human prion diseases occur in infectious, sporadic or genetic forms. The "protein only" hypothesis argues that the key event in the pathogenesis represents the conversion of the normal host protein, PrPc, into its pathogenic isoform PrPSc. Prion diseases have been associated with the accumulation of this abnormally folded protein and its neurotoxic effects. However, it is not known if PrPc loss of function is an important factor since the normal biological function of PrPc, a cell surface-anchored glycoprotein predominantly expressed in neuronal cells, and the cellular processes in which this protein is involved remain obscure. Recently, the human 37 kDa laminin receptor precursor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), was identified as a binding partner for the cellular prion protein in a yeast two-hybrid screen. In order to characterize the possible role of LRP/LR as a cell surface receptor for PrPc, cell culture studies were performed to investigate the cellular localization of PrP and LRP/LR and to analyse the binding and internalization behaviour of PrP depending on the presence of LRP/LR on the cell surface of neuronal and non-neuronal cells. Immunofluorescence analysis of non-permeabilized murine neuroblastoma cells demonstrated that PrP and LRP/LR co-localize on the surface of these cells. In addition, baby hamster kidney (BHK) cells transfected with recombinant Semlik-Forest virus RNAs overexpressed human PrP and human LRP at their cell surface, the latter one orientated as a type II transmembrane protein with its C-terminus outside and its N-terminus inside the cell. Co-localization of both proteins was observed on BHK cells co-transfected with LRP and PrP encoding recombinant SFV RNAs. Cell binding and internalization assays with recombinant human PrP demonstrated the LRP/LR-dependent binding and endocytosis of externally added human PrP. An increased, dose-dependent cell binding of recombinant PrP was demonstrated by BHK cells overexpressing full-length human LRP on their cell surface. Trypsin treatment of the cell surface revealed the LRP dependent internalization of GST-tagged and untagged, glycosylated PrP. In contrast to wild-type LRP, the expression of an LRP mutant lacking its transmembrane domain led to the secretion of this mutant from transfected BHK cells and totally abolished the binding and internalization of exogenous, recombinant PrP. This LRP mutant could function as a decoy recetor in therapy of TSEs. The strict LRP/LR specificity of the PrP binding to neuronal cells was verified by testing the displacement capacity of a series of different antibodies in the LRP-PrP binding reaction. Only LRP and PrP specific antibodies were able to block totally the binding of human GST-fused PrP to N2a and NT2 cells whereas various control antibodies used for competition showed no effect. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of cellular PrP to the 37-kDa/67-kDa LRP/LR. The relationship between the 37-kDa LRP and the 67-kDa high-affinity LR is unknown so far. Both forms were observed in plasma membrane fractions of N2a cells. We conclude from these data that the 37-kDa/67-kDa laminin receptor acts as the main cell surface receptor for PrP. High-level expression and purification of recombinant, glycosylated prion proteins in mammalian cells is essential for a better understanding of the physiological function of PrPc and biochemical processes responsible for prion diseases. Due to the presence of important organelles, membranes and other cellular cofactors which are necessary for the correct processing, trafficking and localization of prion proteins mammalian cell culture systems such as the Semliki-Forest virus (SFV) system allow the synthesis and characterization of wild-type as well as mutant PrP to get a better insight into the biology of these proteins. Therefore, the SFV system was used to generate recombinant highly glycosylated human wild-type and human disease-associated mutant prion proteins as well as FLAG-tagged human and bovine PrP in cultured BHK cells. Both mutated variants, which are related to the human prion diseases fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) reveal proteinase K (PK) resistance, one of the most typical biochemical properties characteristic for the infectious scrapie isoform of the prion protein. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP without any significant differences regarding the cellular distribution and expression level. In addition, FLAG-tagged prion proteins were expressed with high efficiency in BHK cells showing the typical glycosylation pattern allowing the rapid and simple purification via anti-FLAG antibody chromatography. PrP dimers could play an essential role in the PrPc to PrPSc conversion process and might be involved in PrP interspecies transmission. Recently, crystallization of the prion protein in a dimeric form was reported. Size exclusion chromatography showed that native soluble homogeneous FLAG tagged prion proteins from hamster, man and cattle expressed in the baculovirus system were predominantly dimeric. The PrP/PrP interaction was confirmed in rec. SFV-RNA transfected BHK cells co-expressing FLAG and oligohistidine tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. The identification of the 37-kDa/67-kDa laminin receptor as the receptor for the cellular prion protein might represent an important step for a better understanding of the molecular biology of prion diseases and might lead to the development of powerful therapeutics such as LRP/LR specific antibodies for the treatment of these unconventional diseases.

Prion diseases are rare but fatal neurodegenerative diseases which occur both in humans and mammals caused by the prion protein (PrP) which is well conserved among the species. In this thesis the biochemical properties and the function of prion protein were investiagted using different methods. The oligomerisation state of the prion protein analysed by size exclusion chromatography revealed that the prion protein is dimeric under native conditions. This was proven in the yeast two-hybrid system followed by the identification of two interaction domains. The influence of mutations and polymorphisms within the prion gene was investigated in the yeast two-hybrid system. This method is also an useful tool for the investigation of the species barrier. To determine the role of dimeric prion proteins on the scrapie prion protein formation a covalently-linked PrP dimer was constructed and expressed in yeast Pichia pastoris. The protein was expressed as a glycosylated, proteinase K sensitive protein which is transported to the plasma membrane of yeast cells. Recently, the 37-kDa/67-kDa laminin receptor LRP/LR was identified as the receptor for cellular PrP. Besides the in vitro interaction of PrP and LRP the binding domains on PrP and LRP were mapped in the yeast two-hybrid system. In addition, cell binding assays revealed a second HSPG-dependent binding domain leading to a comprehensive model of the PrP/LRP complex on the cell surface. The binding of heparan sulfates to the prion protein was investigated and binding domains located on the N-terminus of the prion protein were characterized using biosensor and ELISA methodology. The prion-like protein Doppel (Dpl) shows neither an interaction with PrP nor with LRP, the receptor for the cellular prion protein, in the yeast two-hybrid system, but is well expressed in yeast cells. The tyrosine kinase Fyn, activated by PrP in neuronal cells, interact directly with PrP in the yeast two-hybrid system whereas LRP failed to interact with Fyn.