Cytosolic Prion Protein Research Articles

Protective role of cytosolic prion protein against virus infection in prion-infected cells.

Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections.

Cross-seeding by prion protein inactivates TDP-43.

A common pathological denominator of various neurodegenerative diseases is the accumulation of protein aggregates. Neurotoxic effects are caused by a loss of the physiological activity of the aggregating protein and/or a gain of toxic function of the misfolded protein conformers. In transmissible spongiform encephalopathies or prion diseases, neurodegeneration is caused by aberrantly folded isoforms of the prion protein (PrP). However, it is poorly understood how pathogenic PrP conformers interfere with neuronal viability. Employing in vitro approaches, cell culture, animal models and patients' brain samples, we show that misfolded PrP can induce aggregation and inactivation of TAR DNA-binding protein-43 (TDP-43). Purified PrP aggregates interact with TDP-43 in vitro and in cells and induce the conversion of soluble TDP-43 into non-dynamic protein assemblies. Similarly, mislocalized PrP conformers in the cytosol bind to and sequester TDP-43 in cytosolic aggregates. As a consequence, TDP-43-dependent splicing activity in the nucleus is significantly decreased, leading to altered protein expression in cells with cytosolic PrP aggregates. Finally, we present evidence for cytosolic TDP-43 aggregates in neurons of transgenic flies expressing mammalian PrP and Creutzfeldt-Jakob disease patients. Our study identified a novel mechanism of how aberrant PrP conformers impair physiological pathways by cross-seeding.

Aberrant SENP1-SUMO-Sirt3 Signaling Causes the Disturbances of Mitochondrial Deacetylation and Oxidative Phosphorylation in Prion-Infected Animal and Cell Models

Post-translational modifications of proteins, such as acetylation and SUMOylation, play important roles in regulation of protein functions and pathophysiology of different diseases including neurodegenerative diseases. Our previous studies have identified aberrant acetylation profiles and reduced deacetylases Sirt3 and Sirt1 in the brains of prion-infected mouse models. In this study, we have found that the levels of acetylated forms of AceCS2 and LCAD, the key enzymes regulating lipid metabolism, CS and IHD2, the key enzymes regulating complete oxidative metabolism, GDH, the key enzyme regulating the oxidative decomposition of glutamate into the tricarboxylic acid (TCA) cycle, and NDUFA9, the essential component in the complex I of respiratory chain activity, were significantly upregulated in the prion-infected animal and cell models, along with the decrease of Sirt3 activity and mitochondrial cytochrome c oxidase activity. Meanwhile, the increases of SUMO1 modifications and SUMO1-Sirt3 and decrease of SENP1 were identified in the brains and the cultured cells with prion infections. Removal of prion propagation in the cultured cells partially, but significantly, reversed the aberrant situations. Moreover, similar abnormal phenomena were also observed in the cultured 293 T cells transiently expressing cytosolic form PrP (Cyto-PrP), including decreased SENP1, increased SUMO1, decreased Sirt3 activity, increased acetylated forms of the key enzymes, and decreased cytochrome c oxidase activity. Attenuation of the accumulation of Cyto-PrP by co-expression of the p62 protein sufficiently diminished those abnormalities. The data here strongly indicate that deposits of prions in brains or accumulations of Cyto-PrP in cells trigger dysregulation of the SENP1-SUMO1-Sirt pathway and subsequently induce aberrant mitochondrial deacetylation and the mitochondrial respiratory chain.

Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease.

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. The cellular and molecular mechanisms of prion-induced neurotoxicity remain undefined. Understanding these processes will underpin therapeutic and control strategies for human and animal prion diseases, respectively. Prion diseases are difficult to study in their natural hosts and require the use of tractable animal models. Here we used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes we observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, our data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology.

Cytosolic aggregates in presence of non-translocated proteins perturb endoplasmic reticulum structure and dynamics.

Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non-translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non-translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three-way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins-reticulons, and altered distribution of those associated with the three-way ER junctions-Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non-translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven-pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non-translocated precursor proteins-their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.

Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis

Unfolded protein response (UPR) is an adaptive reaction for cells to reduce endoplasmic reticulum (ER) stress. In many types of cancers, such as lung cancer and pancreatic cancer, cancer cells may harness ER stress to facilitate their survival and growth. Prion protein (PrP) is a glycosylated cell surface protein that has been shown to be up-regulated in many cancer cells. Since PrP is a protein prone to misfolding, ER stress can result in under-glycosylated PrP, which in turn may activate ER stress. To assess whether ER stress leads to the production of under-glycosylated PrP and whether under-glycosylated PrP may contribute to ER stress thus leading to cancer cell apoptosis, we treated different cancer cells with brefeldin A (BFA), thapsigargin (Thps), and tunicamycin (TM). We found that although BFA, Thps, and TM treatment activated UPR, only ATF4 was consistently activated by these reagents, but not other branches of ER stress. However, the canonical PERK-eIF2α-ATF4 did not account for the observed activation of ATF4 in lung cancer cells. In addition, BFA, but neither Thps nor TM, significantly stimulated the expression of cytosolic PrP. Finally, we found that the levels of PrP contributed to anti-apoptosis activity of BFA-induced cancer cell death. Thus, the pathway of BFA-induced persistent ER stress may be targeted for lung and pancreatic cancer treatment.

PrP-containing aggresomes are cytosolic components of an ER quality control mechanism.

Limited detoxification capacity often directs aggregation-prone, potentially hazardous, misfolded proteins to be deposited in designated cytosolic compartments known as 'aggresomes'. The roles of aggresomes as cellular quality control centers, and the cellular origin of the deposits contained within these structures, remain to be characterized. Here, we utilized the observation that the prion protein (PrP, also known as PRNP) accumulates in aggresomes following the inhibition of folding chaperones, members of the cyclophilin family, to address these questions. We found that misfolded PrP molecules must pass through the endoplasmic reticulum (ER) in order to be deposited in aggresomes, that the Golgi plays no role in this process and that cytosolic PrP species are not deposited in pre-existing aggresomes. Prior to their deposition in the aggresome, PrP molecules lose the ER localization signal and have to acquire a GPI anchor. Our discoveries indicate that PrP aggresomes are cytosolic overflow deposition centers for the ER quality control mechanisms and highlight the importance of these structures for the maintenance of protein homeostasis within the ER.

Stabilization of microtubular cytoskeleton protects neurons from toxicity of N-terminal fragment of cytosolic prion protein

Prion protein (PrP) mislocalized in the cytosol has been presumed to be the toxic entity responsible for the neurodegenerative process in transmissible spongiform encephalopathies (TSE), also called prion diseases. The mechanism underlying the neurotoxicity of cytosolic PrP (cytoPrP) remains, however, unresolved. In this study we analyze toxic effects of the cell-penetrating PrP fragment, PrP1–30 — encompassing residues responsible for binding and aggregation of tubulin. We have found that intracellularly localized PrP1-30 disassembles microtubular cytoskeleton of primary neurons, which leads to the loss of neurites and, eventually, necrotic cell death. Accordingly, stabilization of microtubules by taxol reduced deleterious effects of cytosolic PrP1–30. Furthermore, we have demonstrated that decreased phosphorylation level of microtubule-associated proteins (MAPs), which also increases stability of microtubular cytoskeleton, protects neurons from the toxic effects of PrP1–30. CHIR98014 and LiCl — inhibitors of glycogen synthase kinase 3 (GSK-3), a major kinase responsible for phosphorylation of MAPs, inhibited PrP1-30-induced disruption of microtubular cytoskeleton and increased viability of peptide-treated neurons. We have also shown that the N-terminal fragment of cytoPrP may cause the loss of dendritic spines. PrP1–30-induced changes at the level of spines have also been prevented by stabilization of microtubules by taxol as well as LiCl. These observations indicate that the neurotoxicity of cytoPrP is tightly linked to the disruption of microtubular cytoskeleton. Importantly, this study implies that lithium, the commonly used mood stabilizer, may be a promising therapeutic agent in TSE, particularly in case of the disease forms associated with accumulation of cytoPrP.

Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila.

Prion diseases are fatal transmissible neurodegenerative diseases of various mammalian species. Central to these conditions is the conversion of the normal host prion protein PrP(C) into the abnormal prion conformer PrP(Sc). Mature PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol anchor, whereas during biosynthesis and metabolism cytosolic and secreted forms of the protein may arise. The role of topological PrP(C) variants in the mechanism of prion formation and prion-induced neurotoxicity during prion disease remains undefined. In the present study we investigated whether Drosophila transgenic for ovine PrP targeted to the plasma membrane, to the cytosol or for secretion, could produce transmissible toxicity following exposure to exogenous ovine prions. Although all three topological variants of PrP were efficiently expressed in Drosophila, cytosolic PrP was conformationally distinct and required denaturation before recognition by immunobiochemical methods. Adult Drosophila transgenic for pan neuronally expressed ovine PrP targeted to the plasma membrane, to the cytosol or for secretion exhibited a decreased locomotor activity after exposure at the larval stage to ovine prions. Proteinase K-resistant PrP(Sc) was detected by protein misfolding cyclic amplification in prion-exposed Drosophila transgenic for membrane-targeted PrP. Significantly, head homogenate from all three variants of prion-exposed PrP transgenic Drosophila induced a decreased locomotor activity when transmitted to PrP recipient flies. Drosophila transgenic for PrP targeted for secretion exhibited a spontaneous locomotor defect in the absence of prion exposure that was transmissible in PrP transgenic flies. Our data are consistent with the formation of transmissible prions in PrP transgenic Drosophila.

Cytosolic PrP can participate in prion-mediated toxicity.

Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. Importance: During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and prion formation is undefined. Here we generated Drosophila transgenic for cytosolic PrP expression in order to investigate its toxic potential in the absence or presence of exogenous prions. Cytosolic ovine PrP expressed in Drosophila was not overtly detrimental to the flies. However, cytosolic PrP transgenic Drosophila exposed to ovine scrapie showed a toxic phenotype absent from similar flies exposed to scrapie-free material. Significantly, the scrapie-induced toxic phenotype in cytosolic transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that cytosolic PrP can participate in the generation of transmissible prion-induced toxicity and highlight the utility of Drosophila as a model of mammalian prion disease.

Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein.

Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.

Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP(C)) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.

Molecular Interaction of TPPP with PrP Antagonized the CytoPrP-Induced Disruption of Microtubule Structures and Cytotoxicity

BackgroundTubulin polymerization promoting protein/p25 (TPPP/p25), known as a microtubule-associated protein (MAP), is a brain-specific unstructured protein with a physiological function of stabilizing cellular microtubular ultrastructures. Whether TPPP involves in the normal functions of PrP or the pathogenesis of prion disease remains unknown. Here, we proposed the data that TPPP formed molecular complex with PrP. We also investigated its influence on the aggregation of PrP and fibrillization of PrP106–126 in vitro, its antagonization against the disruption of microtubule structures and cytotoxicity of cytosolic PrP in cells, and its alternation in the brains of scrapie-infected experimental hamsters.Methodology/Principal FindingsUsing pull-down and immunoprecipitation assays, distinct molecular interaction between TPPP and PrP were identified and the segment of TPPP spanning residues 100–219 and the segment of PrP spanning residues 106–126 were mapped as the regions responsible for protein interaction. Sedimentation experiments found that TPPP increased the aggregation of full-length recombinant PrP (PrP23–231) in vitro. Transmission electron microscopy and Thioflavin T (ThT) assays showed that TPPP enhanced fibril formation of synthetic peptide PrP106–126 in vitro. Expression of TPPP in the cultured cells did not obviously change the microtubule networks observed by a tubulin-specific immunofluorescent assay and cell growth features measured by CCK8 tests, but significantly antagonized the disruption of microtubule structures and rescued the cytotoxicity caused by the accumulation of cytosolic PrP (CytoPrP). Furthermore, Western blots identified that the levels of the endogenous TPPP in the brains of scrapie-infected experimental hamsters were significantly reduced.Conclusion/SignificanceThose data highlight TPPP may work as a protective factor for cells against the damage effects of the accumulation of abnormal forms of PrPs, besides its function as an agent for dynamic stabilization of microtubular ultrastructures.

Expression of Mutant or Cytosolic PrP in Transgenic Mice and Cells Is Not Associated with Endoplasmic Reticulum Stress or Proteasome Dysfunction

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the UbG76V-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases.

Cytosolic aggregates perturb the degradation of nontranslocated secretory and membrane proteins

A wide range of diseases are associated with the accumulation of cytosolic protein aggregates. The effects of these aggregates on various aspects of normal cellular protein homeostasis remain to be determined. Here we find that cytosolic aggregates, without necessarily disrupting proteasome function, can markedly delay the normally rapid degradation of nontranslocated secretory and membrane protein precursors. In the case of mammalian prion protein (PrP), the nontranslocated fraction is recruited into preexisting aggregates before its triage for degradation. This recruitment permits the growth and persistence of cytosolic PrP aggregates, explaining their apparent "self-conversion" seen in earlier studies of transient proteasome inhibition. For other proteins, the aggregate-mediated delay in precursor degradation led to aggregation and/or soluble residence in the cytosol, often causing aberrant cellular morphology. Remarkably, improving signal sequence efficiency mitigated these effects of aggregates. These observations identify a previously unappreciated consequence of cytosolic aggregates for nontranslocated secretory and membrane proteins, a minor but potentially disruptive population the rapid disposal of which is critical to maintaining cellular homeostasis.

Cell Type-Specific Neuroprotective Activity of Untranslocated Prion Protein

BackgroundA key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.Principal FindingsMild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.SignificanceThese results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

Cytosolic PrP Induces Apoptosis of Cell by Disrupting Microtubule Assembly

Prion protein (PrP) is able to bind with tubulin and to interfere with the formation of microtubule. To investigate the influence of accumulation of cytosolic PrP in cytoplasm on microtubule, plasmid pcDNA3.1-PrP23-230 expressing human PrP23-230 was introduced into HeLa cells. Immunoprecipitation assays identified the molecular interaction between cytosolic PrP and cellular tubulin. Confocal microscopy showed the co-localization of the expressed cytosolic PrP with tubulin in cytoplasm. Immunofluorescent assays of tubulin illustrated remarkable disruption of microtubular structures in the cells accumulated with cytosolic PrP. Meanwhile, the expressed cytosolic PrP significantly reduced cell viability and induced cell apoptosis. The amounts of microtubule protein in the cells expressing cytosolic PrP were decreased. Moreover, the levels of endogenous tubulin in the brain tissues of scrapie-infected hamsters were significantly lower than that of normal one. It highlights a close linkage between disruption of microtubule framework and cell death caused by abnormal presence of cellular PrP in cytoplasm. The association of apoptosis with microtubule-disrupting activity caused by cytosolic PrP may further provide insight into the unresolved biological function of PrP in the neurons.

Pro-prion Binds Filamin A, Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.

A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cell Lines

The development of antibodies effective in crossing the blood brain barrier (BBB), capable of accessing the cytosol of affected cells and with higher affinity for PrPSc would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the “proof-of-principle” for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrPC. In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrPSc replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means.