Poster Session PPo6: Functions and Cell Biology of PrP

PPo6-1: From Agent to Disease: From Macrocyclic Lactones to TSEs

Andreas Becker

Interuniversity College for Health and Development Graz/Castle of Seggau; Austria

Key words: Alzheimer’s disease, BSE, biological function, chronic toxicity, GABA-receptor, macrocyclic lactones, prion, protein-only-hypothesis, scrapie, TSE

Transmissible spongiforme encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases of genetic origin. The mechanism of pathogenicity consists in the formation of prions (prion protein scrapie; PrP^Sc) which reproduce themselves in a chain reaction by conformational change from cellular prion protein (PrP^C). Consequently, an important evolutionary mechanism in the development of cellular formation is revived here: the optimization of the efficiency of a locally limited tissue by a targeted destruction of cells. This is the original biological function of prions.

The iatrogenic transmission of prions is not an infection, just as Alzheimer’s disease is not contagious by experimental transmission of tau proteins. The discovery of the biological function of the PrP^Sc-PrP^C-system questions the validity of the “protein only” hypothesis.

Besides copper deficiency, oversupply of manganese and oxidative stress, chronic intoxication with macrocyclic lactones (ML) may induce conformational change of PrP^C.

In Great Britain, several factors have contributed to the largest pharmaceutical scandal (“apocalypse cow”) since the advent of Contergan, among them the marketing introduction of ML Ivermectin (1982), errors of pharmacological registration by the European Medicines Agency (EMEA), the underestimation of the residual situation of ML, the compulsory warble fly eradication scheme using phosmet, the production of meat and bone meal without extraction of fat, the use of animal fats in milk replacers as well as the incorrect interpretation of prion tests. In sum, evidence suggests that registration of modern ML, such as Eprinomectin and Moxidectin, might be the cause for the atypical forms of BSE and scrapie.

PPo6-2: Abnormal Signaling Mediated by Laminin in Neurons Expressing Mutant Prion Proteins

Cleiton F. Machado,^1,2 Flavio H. Beraldo,¹ Dominique Bourgeon¹ and Vilma R. Martins¹

¹Ludwig Institute for Cancer Research; ²Centro Internacional de Pesquisa e Ensino do Hospital A.C; Camargo, São Paulo Brazil

Key words: prion protein, laminin, mutant

Prion protein (PrP^C) is a glycosylphosphatidylinositol anchored protein predominantly expressed at the surface of neuronal and glial cells. Mutations in the PrP^C gene are involved with genetic forms of prion diseases. Indeed, these mutations represent relevant tools to evaluate the PrP^C loss-of-function within these disease forms. Some PrP^C functions have been associated with its ability to bind specific cellular proteins. We have previously demonstrated that PrP^C region between aminoacids 173 and 182 binds to laminin gamma1 chain, promoting neuritogenesis and memory formation. Three PrP^C mutations, associated with genetic Creutzfeldt-Jakob disease and Fatal Familial Insomnia, were mapped within the PrP^C binding site for laminin. In the present work, cDNAs coding wild-type (WT) mouse PrP^C and mutated mouse PrP^C, equivalent to human proteins, were expressed in PrP^C-null immortalized embryonic mouse neurons. The expression of PrP^C WT (128M) and PrP^C mutants 177N, 179I and 182A was confirmed by western blot, immunofluorescence and flow cytometry. Cells expressing mutant proteins 177N, 179I and 182A presented growth impairment when compared to cells expressing PrP^C WT. Moreover, the expression of 128M in PrP^C-null cells rescued calcium signaling responses mediated by PrP^C-laminin gamma1 peptide interaction. Neurons expressing the mutated PrP^C 179I, presented 30.5 ± 1.1% lower intracellular calcium levels upon laminin gamma1 peptide treatment than those expressing 128M. These data show that mutations on PrP^C can affect protein functions and suggest that the PrP^C loss-of-function may be relevant in the pathogenesis of genetic prion diseases.

PPo6-3: PrP^C Localizes to the Nuclear Lamina of Pancreatic Islet and Brain Cells and Interacts with Nuclear Proteins

Alexander Strom,¹ Gen-Sheng Wang,¹ David J. Picketts,² Rudolph Reimer,³ Andreas W. Stuke⁴ and Fraser W. Scott^1,2

¹Chronic Disease Program; ²Regenerative Medicine Program; Ottawa Hospital Research Institute; Ottawa, Ontario Canada; ³Electron Microscopy and Micro-Technology Group; Heinrich-Pette-Institute; Hamburg, Germany; ⁴Infection Biology Department; German Primate Centre; Göttingen, Germany

Key words: glucoregulation, cellular prion protein, pancreatic islets, hisone H3, lamin B1, nucleus

Cellular prion protein (PrP^C) is best known as the precursor of PrP^Sc (Sc-scrapie) that leads to transmissible spongiform encephalopathies. Several cellular processes have been purported for this protein, but remarkably little is known of the cellular function of the PrP^C. In a recent study we demonstrated that long term hyperglycaemia was associated with β-cell specific changes in PrPC expression and aggregation (Strom et al. Lab Invest 2007; 87:139–49). These findings suggested that PrP^C could play a direct role in β-cell function associated with blood glucose regulation. Here we demonstrate that mice lacking PrPC have a delayed response to hyperglycaemia. Our data show that the impaired blood glucose regulation in PrP knock-out mice does not result from reduced β-cell mass, insulin deficiency or from insulin resistance and suggest the production of insulin with impaired bioactivity. Furthermore, we show for the first time in vivo that PrP^C is highly abundant in the nuclear lamina of endocrine and neuronal cells where it interacts with lamin B1 and histone H3 raising the possibility that PrP^C is involved in transcriptional regulation. In conclusion, we demonstrate here that PrP^C is a novel target molecule that is required for proper response of β-cells to hyperglycaemia. A further understanding of the role of PrP^C in regulating islet cell function will provide valuable insight for blood glucose regulation. In addition, the identification of PrP^C as a nuclear protein provides new prospects in the elucidation of molecular mechanisms leading to neurodegeneration in prion diseases.

PPo6-4: Prion Protein is a Key Determinant of Alcohol Sensitivity through the Modulation of N-methyl-D-aspartate Receptor (Nmdar) Activity

Baptiste Ménard,¹ Alice Guyon,¹ Vincent Béringue,² Nicole Zsürger,¹ Agnès Petit-Paitel¹ and Joëlle Chabry¹

¹Centre National de la Recherche Scientifique (CNRS); Institut de Pharmacologie Moléculaire et Cellulaire; Valbonne, France; ²Institut National de la Recherche Agronomique (INRA); UR892; Virologie et Immunologie Moléculaires; Jouy-en-Josas, France

Key words: prion, physiopathological function, NMDA receptors, alcohol sensitivity, phosphorylation, intra-cellular signaling, electrophysiology

The cellular prion protein (PrPc) is absolutely required for the development of prion diseases; nevertheless its physiological functions in the central nervous system remain elusive. Using a combination of behavioral, electrophysiological and biochemical approaches on transgenic mouse models, we provide strong evidence for a novel physiological role of PrPc at synaptic level. Indeed, PrP knock-out mice presented a greater sensitivity to the sedative effects of ethanol compared to wild-type control mice. Conversely, mice overexpressing mouse, human or hamster PrP sequences were less sensitive than wild-type to ethanol-induced sedative effects. Interestingly, prion-infected mice present also an altered response to ethanol-induced sedation as soon as 12 weeks after inoculation. We show that PrPc is required to induce acute tolerance to ethanol by modulating the N-methyl-D-aspartate receptor activity. This effect requires the activation of a Src-protein tyrosine kinase-dependent intracellular signaling pathway. Together, our results reinforce the concept of a crucial role of PrPc in synaptic function.

PPo6-5: Transcriptomic Analysis Highlights Time-specific Embryonic Adaptation of Mice to the Lack of PrP

Manal Khalife,¹ Rachel Young,¹ Sead Chadi,¹ Sandrine Le Guillou,¹ Lucas Lefevre,¹ Gaëlle Tilly,¹ Frédérique Bitton,² Marie-Laure Martin-Magniette,^2,3 Ludivine Soubigou-Taconnat,² Sandrine Balzergue,² Marthe Vilotte,¹ Bruno Passet,¹ Vincent Béringue,⁴ Jean-Pierre Renou,² Fabienne Le Provost,¹ Hubert Laude⁴ and Jean-Luc Vilotte¹

¹INRA; UMR1313; Génétique Animale et Biologie Intégrative; Jouy-en-Josas, France; ²UMR INRA 1165 CNRS 8114; Recherche en Génomique Végétale; UEVE; Evry Cedex, France; ³UMR AgroParisTech/INRA Mathématique et Informatique Appliquées 518; Paris, France; ⁴INRA; UR892; Virologie Immunologie Moléculaires; Jouy-en-Josas, France

Key words: prion, mouse, transcriptome, knock out, embryo

The physiological function of the PrP remains largely elusive. Its invalidation does not affect mouse survival and induces subtle phenotypes. To potentially assess this conundrum, we first comparatively analyzed the adult brain transcriptome of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic (Zürich PrP^0/0 mice) or adult stages (NFH-Cre-Lox mice). Only subtle differences could be evidenced in the adult brains following microarray and QPCR analyses. When performed at an early adult stage, neuronal Prnp disruption appeared to sequentially induce an oxidative stress response and a nervous system remodeling, but it involved a limited number of only slightly modified genes. In sharp contrast, analysis at early embryonic stages, 7.5 and 8.5 dpc, just after the suspected normal time set of the Prnp locus activation, led to a transient perturbation of the transcriptome involving a larger number of genes and pointing to potential pathways related to the PrP physiological function. Overall, our data suggests an early adaptation of the mouse to the potentially detrimental lack of PrP during embryogenesis while its presence is less influential or redundant at later developmental stages.

PPo6-6: Mammalian PrP Promotes Prion Formation in Yeast

Yury O. Chernoff,¹ Meng Sun,¹ Carmen Krammer² and Ina Vorberg³

¹School of Biology and Institute for Bioengineering and Bioscience; Georgia Institute of Technology; Atlanta, Georgia USA; ²Department of Biochemistry; Northwestern University; Evanston, Illinois USA; and ³Deutsches Zentrum für Neurodegenerative Erkrankungen; Bonn, Germany

Key words: aggregation, prion formation, PrP, Sup35, yeast

Self-perpetuating (prion) isoform of the mammalian prion protein (PrP) is associated with transmissible neurodegenerative diseases, while yeast prions control cytoplasmically heritable traits. Yeast and mammalian prion proteins are not homologous to each other. The N-terminal region (Sup35N, or prion domain) of the yeast translational termination factor Sup35 (eRF3) is responsible for the formation and propagation of the prion isoform of this protein. Overproduction of Sup35 or Sup35N induces de novo formation of the Sup35 prion. Such an induction is usually efficient only in the presence of a prion isoform of another protein, for example Rnq1. We have shown that fusion of Sup35N to mammalian PrP promotes de novo induction of the Sup35 prions in the absence of Rnq1 prion or any other known prions. Thus, fusion to PrP enables a chimeric protein to nucleate new prions even in the absence of pre-existing nuclei. Some effects of PrP are counteracted by the middle region of Sup35 (Sup35M) that is rich in charged residues. Differences in prion-inducing abilities coincide with the differences in cytologically detectable aggregation patterns of the Sup35N-PrP and Sup35NM-PrP constructs. Our data establish a yeast-based assay for studying the prion-related properties of mammalian PrP.

Acknowledgements

Supported by grant R01GM58763 from NIH.

PPo6-7: The Cellular Prion Protein Contributes to the Sprouting of Neurites Along Neuronal Differentiation by Controlling Actin Organization and Focal Adhesion Site Dynamics

Damien Loubet,¹ Caroline Dakowski,¹ Elodie Pradines,¹ Sophie Bernard,² Callebert Jacques,³ Mathéa Piétri,¹ Sophie Mouillet-Richard,¹ Launay Jean-Marie,^3,4 Odile Kellermann¹ and Benoît Schneider¹

¹INSERM U747; Université Paris Descartes; Laboratoire «Cellules Souches, Signalisation et Prions»; Paris, France; ²CNRS UPR 2228; Université Paris Descartes 45 rue des Saints Pères; Paris, France; ³Service de Biochimie; Hôpital Lariboisière; AP-HP; ⁴Pharma Research Department; Hoffmann La Roche AG; Basel, Switzerland

Key words: cellular prion protein, neuritogenesis focal adhesions, actin neuronal differentiation

Background. The sprouting of neurites represents a very early stage in the implementation of the neuronal phenotype and relies on localized actin network reorganization and focal adhesion site turn-over. While increasing data indicate that PrPC takes part to neurite outgrowth, the underlying mechanisms remain elusive.

Objectives. Our goal was (I) to assess the impact of PrPC silencing in neuronal progenitors on actin architecture, focal adhesion site dynamics and neuritogenesis and (II) to define how PrPC contributes to neuronal polarity acquisition.

Results. Our data reveal that PrPC depletion in 1C11 precursor cells impairs neuritogenesis. This relates to actin disorganization, increased actin stress fibre stability and altered Focal Adhesion turn-over. PrPC silencing promotes fibronectin overexpression and Beta1 integrin clustering. Beta1 integrin activation triggers the sustained recruitment of the RhoA-Rock-LimK-Cofilin signalling pathway, which hampers localized actin reorganization in PrPnull-1C11 cells and the initial sprouting of neurites along bioaminergic differentiations.

Discussion. We substantiate that PrPC contributes to the sprouting of neurites along neuronal differentiation of 1C11 precursor cells. By modulating Beta1 integrin interactions with fibronectin, PrPC controls actin architecture and focal adhesion site dynamics necessary to neuritogenesis.

Methods. Our study is based on RT-PCR, western-blot, Atomic Force Microscopy and immunofluorescence experiments. We take advantage of the neuroectodermal 1C11 cell line able to differentiate into serotonergic or noradrenergic neuronal cells. 1C11 precursors and their neuronal progenies endogenously express PrPC at a similar level. We also benefit from 1C11-derived PrP-depleted cells (PrPnull-1C11 cells) obtained through a siRNA-based strategy.

PPo6-8: The Toxic Effect of a Mutant Prion Protein is Cell-autonomous, but can be Suppressed in trans by Wild-type Prion Protein

Emiliano Biasini, Jessie A. Turnbaugh, Tania Massignan and David A. Harris

Department of Biochemistry; Boston University School of Medicine; Boston, MA USA

Key words: PrP function, mutant PrP toxicity, neural stem cells

Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrPC) in this process, remain enigmatic. A window into the normal function of PrPC, and how it can be corrupted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The most toxic of these mutants, δ105–125 (designated δCR) produces a neonatal lethal phenotype when expressed in transgenic mice lacking endogenous PrP.

In a previous study, we reported that cells expressing δCR PrP are hyper-sensitive to the toxic effects of two antibiotics (G418 and Zeocin) commonly used for selection of stable transformants. Importantly, the drug sensitizing effect was suppressed by co-expression of wild type (WT) PrP, similar to the rescue of the neurodegenerative phenotype observed in transgenic mice.

Here we characterized this phenomenon in neural stem cells (NSCs) derived from transgenic mice expressing δCR PrP. Once differentiated, these cells can give rise to mixed cultures of neurons, astrocytes and oligondendrocytes. We conducted experiments in which EGFP-labeled and unlabeled NSCs were mixed, differentiated, and treated with G418 or Zeocin to study both the hypersensitivity effect conferred by the mutant protein and the rescuing activity of the WT form.

We report the surprising observation that while the δCR-dependent drug hypersensitivity is cell autonomous, the WT-dependent rescuing activity can be exerted in trans by nearby cells. These results provide important insights into how δCR PrP subverts a normal physiological function of PrPC, and the cellular mechanisms underlying the rescuing process.

PPo6-9: Rab GDP Dissociation Inhibitor Alpha (GDI) Regulates the Trafficking of Mutant Prion Proteins to the Cell Surface

Tania Massignan,¹ Emiliano Biasini,¹ Valentina Bonetto² and David A. Harris¹

¹Department of Biochemistry, Boston University School of Medicine; Boston, MA USA; ²Dulbecco Telethon Institute and Department of Molecular Biochemistry and Pharmacology; Istituto di Ricerche Farmacologiche Mario Negri; Milano, Italy

Key words: mutant prion protein, Rab GDP dissociation inhibitor alpha, protein trafficking, protein aggregation

Inherited prion diseases are associated with mutations in the prion protein (PrP) gene on chromosome 20. These mutations are thought to favour the conformational conversion of PrP into a misfolded, pathogenic isoform. It was previously shown that in primary neurons and transfected cell lines mutant PrP molecules misfold soon after synthesis in the endoplasmic reticulum (ER) and are delayed in their trafficking to the cell membrane.

Previous studies aimed at defining the cellular pathways affected by the expression of a mutant PrP (D178N/M129), highlighted a potentially toxic feedback involving overexpression of Rab GDP dissociation inhibitor alpha (GDI), which could lead to alterations of protein trafficking between the Golgi and the plasma membrane, and induce further accumulation of mutant PrP in the exocytic pathway.

Here we provide additional characterization of the role of GDI in regulating the aggregation and trafficking of multiple mutants of PrP. Moreover, we describe the effect of modulating GDI expression in a novel drug-based cell assay for testing the activity of neurotoxic PrP mutants.

We are currently testing the hypothesis that GDI-mediated intracellular retention of mutant proteins could act as a defensive cell mechanism which prevents aberrantly folded species to reach the cell surface, where they may exert their toxic effect. Thus, restoring the correct delivery of mutant PrPs to the cell membrane by decreasing the level of GDI could be detrimental to cell viability instead of beneficial.

PPo6-10: Cellular Prion Protein Controls the mRNA Expression of Pluripotency Genes and Differentiation in Mice Embryonic Stem Cells

Alberto Miranda, Miguel Ángel Ramírez, Eva Pericuesta and Alfonso Gutiérrez-Adán

Prion protein (PrPc) is a glycoprotein involved in the pathogenesis of a small number of diseases called Transmissible Spongiform Encephalopaties (TSEs) that mainly affect the central nervous system. The main causes of TSEs signs are proposed to be due to gain or loss of this protein’s functions, making more important the study of the processes where PrPc is involved. In this work, we have compared two PrPc-null (KO) stem cell lines to a WT stem cell line during differentiation. The first feature observed was a new population of Embryoid Bodies (EBs) that appears in the KO lines and expresses several Primordial Germ Cell (PGC) markers until day 13 of differentiation, while the WT line doesn’t. We then compared RNA expression pattern of some developmental important genes between the different cell lines. A PrPc implication in the expression of several pluripotent markers, like Nanog mRNA levels during differentiation to EBs was observed, as well as a clear switch off of Nanog at day 5 in the KO lines. It is contrary to WT line when a maximum PrPc and Nanog mRNA levels are needed at this time. These results were confirmed in the WT line using a specific antibody against PrPc (SHA31) to block the activation or inhibition of putative PrPc pathways. RT-PCR analysis showed a significant variation of certain genes expression, including a downregulation of Nanog, when EBs are treated with the antibody. Taken together all data, this is the first known description of a PrPc relationship with major pluripotency genes

PPo6-11: The PrP-like Shadoo Protein Binds Nucleic Acids

Agnes Lau and David Westaway

Centre for Prions and Protein Folding Diseases; Department of Medicine; University of Alberta; Edmonton, Alberta USA

Prion diseases include Creutzfeldt-Jakob Disease and bovine spongiform encephalopathy. The infectious prion protein PrP^Sc involved in these diseases results from a conformational change of the cellular prion protein (PrPC), which is part of the prion superfamily also including the glycoproteins Shadoo (Sho) and Doppel. Sho is the newest member of the family to be characterized and shares several properties with PrPC. To study the binding properties of Sho, N-terminal fragments Sho25-61 and Sho25-80 were fused to glutathione-S-transferase (GST). In addition, GST fusion proteins bearing N-terminal PrP fragments (either with or without positively charged regions) were synthesized as internal controls. During preliminary affinity chromatography experiments, unexpected differences were immediately apparent between affinity columns containing Sho fusion proteins and control PrP fusion proteins. The material from each column analyzed by silver staining or ethidium bromide staining of SDS-PAGE gels revealed that Sho bound to nucleic acids present within the E. coli cell lysate. Treatment of the lysates with RNAse decreased the level of ethidium bromide staining, indicating that Sho bound RNA. In other studies, salt-washing to strip recombinant Sho of endogenous E. coli nucleic acids also revealed an ability to bind double-stranded DNA. The nucleic acid-binding properties of Shadoo offer a striking parallel to the behaviour of N-terminal PrP fragments, as reported by several research groups. Sho-GST fusion proteins will be useful tools to help further characterize the biochemical attributes of the Shadoo N-terminal region.

PPo6-12: Soluble, Fibrillar Amyloid-beta Oligomers Bind to the Cellular Prion Protein and Reduce its Cell Surface Expression

Jo V. Humphrey and Nigel M. Hooper

Institute of Molecular and Cellular Biology; Faculty of Biological Sciences; University of Leeds; Leeds, UK

Key words: PrPC, amyloid-beta, oligomer, Alzheimer’s disease

The cellular prion protein (PrPC) is a neuronal receptor for the primary neurotoxic assemblies in Alzheimer’s Disease (AD), amyloid-beta (Aβ) oligomers (Lauren et al. Nature 2009; 457:1128–32). Further, PrPC modulates the Aβ oligomer-mediated neurodegeneration and memory deficits in an AD model mouse (Gimbel et al. J Neurosci 2010; 30:6367–74). The cell biology of the Aβ:PrPC interaction therefore warrants urgent investigation. Here, we have used immunofluorescence microscopy and flow cytometry to demonstrate that binding of biotin-labelled Aβ1-42 oligomers to SH-SY5Y human neuroblastoma cells expressing PrPC is 10-fold higher than to untransfected cells that lack PrPC. Atomic force microscopy confirmed the presence of spherical Aβ assemblies of 4–5 nm in diameter with no fibrils or protofibrils in our oligomer preparation. We characterised structurally these Aβ oligomers using two conformation-specific antibodies which recognise mutually exclusive fibrillar (OC⁺) or prefibrillar (A11) backbone epitopes. Dot-blotting revealed that the Aβ oligomers which bind PrPC are fibrillar (OC⁺). Interestingly, fibrillar oligomers correlate strongly with AD severity and are significantly elevated in human AD brain regions of high PrPC expression (Tomic et al. Neurobiol Dis 2009; 35:352–8). Furthermore, we used immunofluorescence microscopy to reveal that incubation of SH-SY5Y cells expressing PrPC with nanomolar concentrations of Aβ oligomers causes a significant (approx. 60%) reduction in cell surface PrPC. We propose that the internalisation of PrPC may be a key event in, or even a requirement for, PrPC-mediated Aβ oligomer neurotoxicity, and that inhibition of PrPC endocytosis may therefore prevent Aβ oligomer-mediated AD neuropathology.

PPo6-13: Focus on the Antiviral Properties of the Cellular Prion Protein PrPC/CD230

Sandrine Alais,^1,4 Gaëlle Georges,^2,4 Clarisse Berlioz-Torrent³ and Pascal Leblanc^1,4

¹INSERM U758; unité de virologie humaine; Ecole Normale Supérieure de Lyon; Lyon, France; ²Unité de Biologie des Infections Virales Emergentes (UBIVE); Lyon, France; ³Institut Cochin; INSERM U1016; CNRS UMR 8104; Université Paris Descarte; Département Biologie Cellulaire et Interactions Hôte-Pathogènes; Laboratoire Interactions Hôte-Virus; Hôpital Cochin; Paris; ⁴IFR 128 Biosciences Lyon-Gerland; Lyon, France

Key words: PrPC, HIV-1, antiviral, restriction factor

The cellular prion protein PrPC/CD230 is a GPI anchor protein strongly conserved among the species and well expressed in cells from the nervous and immune systems. The cellular function of PrPC is still under debate, however, studies realized in vivo and in vitro suggested that PrPC is involved in different normal biological processes. In the last decade, independent studies revealed that PrPC displays antiviral properties affecting the replication of different viruses and especially retroviruses like the Human Immunodeficiency Virus type 1 (HIV-1). In this context, we previously showed that PrPC displays important similarities with HIV-1 Nucleocapsid protein and found that PrPC expression in a human cell line strongly reduced HIV-1 production. Here we found that PrPC antiviral properties are not dependant of HIV-1 factor such as the Envelope glycoprotein, Vpu, Nef and Vpr accessory proteins. Using different PrPC mutants, our data indicated that interaction of PrPC with membranes is essential for this anti-viral function.

Comparison of PrPC anti-HIV-1 properties with those recently found with the GPI anchor restriction factor Tetherin/BST2/CD317, revealed that PrPC strongly enhances Tetherin effect but affects HIV-1 production in a different way.

Using Biochemistry and cell imaging strategies we found that PrPC colocalizes with HIV-1 virus assembly machinery and interacts with the HIV-1 Gag structural protein through the Nucleocapsid and Matrix domains. PrPC was found associated with viral particles and concentrated at the virological synapse. Using RNA interference, our data indicated that HIV-1 replication was 2 fold enhanced in a PrPC knockdown T cell line.

PPo6-14: Behavioral Abnormalities in Prion Protein Knockout Mice

Matthias Schmitz, Catharina Greis, Walter Schulz-Schaeffer, Andre Fischer and Inga Zerr

Key words: prion protein, learning, memory, PrP^0/0 mice

Background. The cellular prion protein is a glycoprotein attached to the outer cell surface of the plasma membrane. However, the exact physiological function of PrPc is not completely understood.

Mouse models in which PrPc gene (Prnp) was ablated have been used in several studies to examine behavior and cognition. First observations on PrPc knockout mice PrP0/0 did not show any important behavioral abnormalities (Bueler et al. 1992). However some recent studies had pointed out some differences between PrP0/0 and wildtype mice (WT). PrP0/0 mice exhibit an altered circadian rhythm and alterations in sleep (Tobler et al. 1996).

Furthermore, locomotor performance, latent learning and long term-memory or synaptic transmission (Martins 2002; Wong et al. 2001) can be influenced by PrPc.

Objectives. Behavioral abnormalities in PrP knockout mice should be investigated by using a battery of tests.

Results. In this study we examined age related behavioral changes in PrP0/0 mice in comparison to WT PrP+/+ mice. Three, nine and twenty four old mice were submitted to a battery of behavior tasks. We detected significant differences between PrP0/0 and WT mice in the Open Field, Elevated Plus Maze and Fear Conditioning. Performing the other tasks, PrP0/0 mice remained unremarkable.

Methods. Male knockout mice homozygous for the disrupted Prnp gene, Prnp null mice (Zrch I, 29B6) produced as previously reported (Bueler et al. 1992). In our behavior study we used a battery of different tasks.

Conclusion. Altogether, our data suggests several behavior abnormalities of PrP0/0 mice, revealing further physiological functions of PrPc.

PPo6-15: Neurotoxic Mutants of the Prion Protein Induce Spontaneous Ion Channel Activity

Isaac H. Solomon,^1,2 James E. Huettner² and David A. Harris ¹

¹Department of Biochemistry; Boston University School of Medicine; Boston, MA USA; ²Department of Cell Biology and Physiology; Washington University School of Medicine; St. Louis, MO USA

Key words: prion, channel, cation, mutation, neurodegeneration, patch clamp

Prion diseases are transmissible neurodegenerative disorders caused by a conformationally altered form of the prion protein (PrP). While much is known about infectious prion propagation, the mechanisms by which neurotoxic forms of PrP kill nerve cells remain poorly understood. Transgenic mice expressing certain deletion mutants of PrP display a spontaneous neurodegenerative phenotype in the absence of aggregated or infectious PrP, providing an opportunity to study the neurotoxicity in the absence of other PrP activities. We have previously shown that cells expressing these PrP deletion mutants are hypersensitive to certain cationic drugs (Massignan et al. J Biol Chem 2010; 285:7752–65). This effect was diminished by depolarization of the cellular membrane potential, hinting at the involvement of ion channels or transporters. We report here that the most toxic of the PrP deletion mutants (δ105–125) induces large, spontaneous ionic currents that can be recorded by patch clamping techniques. These currents appear to be mediated by a relatively non-selective cation channels, and can be silenced either by overexpression of wild-type PrP or by a sulfated glycosaminoglycan. Currents with properties similar to those associated with δ105–125 PrP are induced by PrP molecules carrying several different point mutations in the central region that are associated with familial prion diseases of humans. Our results indicate that the neurotoxicity of some mutant forms of PrP may be attributable to enhanced ion channel activity, and that wild-type PrP possesses a channel-silencing activity. PrP-associated channels could therefore represent novel therapeutic targets for treatment of patients with prion diseases.

PPo6-16: The Polybasic N-terminal Region of the Prion Protein is Essential for its Neuroprotective Activity

Jessie A. Turnbaugh,^1,2 Laura C. Westergard² and David A. Harris¹

Department of Biochemistry; Boston University School of Medicine; Boston, MA USA; ²Department of Cell Biology and Physiology; Washington University School of Medicine; St. Louis, MO USA

Key words: prion, neuroprotective activity, cerebellar granular neruons, transgenic mice

Although a great deal has been learned about PrP^Sc and its role in prion propagation, the normal function of the cellular prion protein (PrPC) has remained mysterious. Several intriguing lines of evidence suggest that PrPC may exert a neuroprotective activity against different sources of cellular stress. One of the clearest demonstrations of such activity is the ability of wild-type PrPC to suppress the phenotype of transgenic (Tg) mice expressing a deleted form of PrP (δ32–134, called F35 PrP), which normally develop a spontaneous neurodegenerative illness characterized by ataxia and massive degeneration of cerebellar granule neurons (CGNs).

In an effort to define the domains of PrP involved in neuroprotection, we have created transgenic mice expressing PrP molecules harboring one of three consecutive, N-terminal deletions: δ23–31, δ23–111, and δ23–134. Each of these transgenic lines were crossed to Tg(F35) mice, and their potential rescuing effect was observed.

We found that Tg mice expressing either the F35 protein alone, or in combination with each of the N-terminal deletion mutants, displayed marked loss of CGNs and become terminally ill at similar timepoints. Conversely, Tg(F35) mice co-expressing similar levels of wild-type PrP remained healthy for more than one year. These results suggest that the N-terminal polybasic residues are essential for the neuroprotective activity of PrP.

PPo6-17: The Role of Glycosylation on the Cell Biology of PrPC

Kayleigh C. Iremonger, Enrico Cancellotti and Jean C. Manson

Roslin institute & R(D)SVS; University of Edinburgh; Edinburgh, Scotland UK

The cellular prion protein (PrPC) is a host glycoprotein that plays an essential role in the transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases. PrPC contains two glycosylation sites that can be variably occupied such that 4 different glycotypes exist in vivo. The significance of the variably glycosylated forms of PrP in the physiological function and in disease is unknown. Glycosylation of PrP has been proposed to play a role in strain determination of disease and targeting to either the central (CNS) of peripheral (PNS) nervous systems.

Gene targeted transgenic mice have been created to selectively abolish the glycosylation sites of PrP, to yield either mono- or un-glycosylated forms of PrPC. These mice are being used to study the expression and cell biology of the different glycotypes in order to understand the significance of the glycotypes in health. Isolation of lipid raft regions of the membrane show that all glycotypes are membrane bound and localised to lipid rafts, however unglycosylated PrP is mainly intracellular, probably associated with the golgi apparatus. All glycotypes have the ability to undergo alpha cleavage but unglycosylated PrP is more prone to cleavage than other glycoforms, which may be due to extended periods in intracellular compartments and exposure to PrP cleavage enzymes.

The differences in the trafficking and cell biology of the glycotypes may represent different functions of PrPC. It may also go towards explaining the different susceptibilities to TSE strains seen in the PrP glycosylation deficient mice.

PPo6-18: Toxicity in Cerebellar Slice Cultures of Murine Prion Protein with a Deletion of Residues 105–125

Ursula Unterberger, Emiliano Biasini and David A. Harris

Department of Biochemistry; Boston University School of Medicine; Boston, MA USA

Key words: mutant prion protein, PrPΔ105–125, PrPΔCR, cerebellar slice cultures, neurotoxicity

To identify sequence domains important for neurotoxic and/or neuroprotective properties of the prion protein (PrP), our lab previously engineered transgenic mice expressing a murine prion protein with a deletion of amino acid residues 105–125, also referred to as PrPΔCR (Li et al. EMBO J 2007). The most striking neuropathological feature in these mice is a severe loss of cerebellar granule neurons, beginning around postnatal day 13 and progressing until the animals die at the age of 25 days on average.

Further dissection of the mechanism of neuronal death in the TgΔCR model requires a suitable in vitro experimental system that enables pharmacological manipulations. However, primary granule neurons from TgΔCR animals, as well as cell lines transfected with PrPΔCR, do not die spontaneously in culture. We therefore established a cerebellar slice culture system that allows us to monitor cell death under conditions where the three-dimensional cytoarchitecture is preserved.

We found that neurons of the granule layer die spontaneously in the slice cultures, as revealed by imaging of propidium iodide uptake, at an age that corresponds to the events observed in vivo, suggesting that environmental factors reproduced in this slice system are crucial for the neurotoxic effect of PrPΔCR. In addition, the cells are hypersensitive to the toxic effects of certain antibiotics, confirming previous observations in cell cultures (Massignan et al. J Biol Chem 2010). These results establish our cerebellar slice culture system as a reliable ex vivo model for investigating the neurotoxic effects of PrPΔCR.

PPo6-19: Role of Lipid Rafts in Prion Protein Metabolism, Studied by Alteration of the Glycosphingolipids Content of Cultured Neurons

L. Botto, D. Cunati, M. Masserini and P. Palestini

Department of Experimental Medicine (DIMS); University of Milano-Bicocca; Monza, Italy

Key words: DRM, prion, gangliosides

The Prion protein is a GPI-anchored protein primarily concentrated in neuronal cells. Under certain conditions its cellular form, PrP^C, can convert into the lethal Prion isoform, PrP^Sc. This conversion occurs in particular regions of the membrane enriched with cholesterol and glycosphingolipids, called lipid rafts. The aim of our project is to establish if the alteration of cell lipid composition can modify the membrane distribution of PrP^C within raft or non-raft regions and to promote the activity of disintegrins (ADAM10/17) upon PrP^C.

Due to this reason, granule cells obtained from the cerebella of 8-days-old SD rats were incubated after 8 days in culture with 2 x 10-6 M GM1 or other gangliosides at 4° or 37°C for 4 hours. Detergent resistant membrane fractions (DRM) preparation was accomplished according to Palestini et al. 2000 and proteins in all gradient fractions were separated and analyzed by EF/WB. Lipids were separated by HPTLC and analyzed by radiochromatoscanner.

The treatment with GM1 at 4°C led to a remarkable enrichment of this ganglioside in DRM and slightly increased the content of PrPC while ADAM10/17 amount decreased in this region. At 37°C GM1 was enriched in DRM and a moderate ganglioside incorporation was detectable in non-rafts membrane regions corresponding to gradient fractions from 8 to 12, were there are inside membranes in addition to plasma membrane. In DRM PrPC and ADAM10/17 amounts decreased. Further experiments with different gangliosides mean to verify whether their major encumbrance can change into a bigger extent proteins distribution within raft and non-raft membrane regions.

PPo6-20: AltPrP: A Novel Cryptic Out-of-frame Polypeptide Encoded by Prnp in Higher Mammals

Benoît Vanderperre, Antanas Staskevicius, Guillaume Tremblay and Xavier Roucou

Université de Sherbrooke; déparment de Biochimie; Sherbrooke, QC Canada

Key words: out-of-frame translation, initiation, mitochondria, ER, stress, proteasome

The use of alternative translation initiation codons within a single mRNA contributes to the generation of protein diversity. We noticed that an out-of-frame initiation codon located in a strong Kozak context is present at bps + 90–92 in the human PrP gene. This new putative open reading frame encodes for a 73 amino acid polypeptide covering the octapeptide repeat region of PrP and is extremely rich in tryptophan, since the PHGGGWGQ motif is changed to LG(A/T)A(S/P)WWW. For clarity purposes, we termed this polypeptide alternative PrP (AltPrP).

In order to assess the normal expression levels of AltPrP, a hemagglutinin (HA) tag was inserted within human PrP cDNA in AltPrP’s reading frame (frame 3) to produce carboxy-tagged AltPrP. This construct, termed PrP(HA), provided an HA signal only if AltPrPHA was expressed. Expression of AltPrPHA in cells transfected with PrP(HA) was confirmed by immunoblot using anti-HA antibodies. Inactivation of the alternative out-of-frame initiation codon abolished expression of AltPrPHA. These results were reproduced with PrP(HA) derived from sheep, cattle and deer PrP cDNAs. Expression of AltPrPHA considerably increased following stresses implicated in prion diseases, such as proteasome inhibition and endoplasmic reticulum stress. Immunofluorescence experiments showed that AltPrP is a mitochondrial protein. Sodium bicarbonate extraction of purified mitochondria demonstrated that AltPrPHA is a membrane integral protein.

We conclude that AltPrP is a novel protein encoded within the PrP mRNA sequence in several species of mammals. The impact of AltPrP on the biology of PrP and the pathogenesis of prion diseases is under investigation.

PPo6-21: The Glycine Rich Region of the Prion Protein Modulates Uptake of Prion Infectivity

A.F. Hill, C. F. Harrison, V.A. Lawson, C.L. MasterS, R. Cappai and K.J. Barnham

University of Melbourne; Parkville, Victoria Australia

Key words: prion, PrP, hydrophobic domain, cell mode

The hydrophobic domain of the prion protein (PrP) is very highly conserved and contains a series of GxxxG motifs which are found in protein transmembrane domains (including the Aβ peptide) acting as sites for protein-protein interactions. As PrP contains more than one GxxxG motif, we have termed this region the glycine rich region (GRR). The PrP GRR contains a series of flexible residues within a region known to undergo significant structural change in prion formation. We generated a panel of prion infectable cell lines expressing PrP containing amino-acid substitutions in the GRR. Conservative mutations within the GRR have a drastic effect on the propagation of prions, demonstrating this region of PrP is a dominant modulating factor of prion infectivity. Reduction in prion infection appears proportional to the decrease in conformational flexibility in the GRR, implying that the PrP conversion requires significant structural change within this region. Additional mutations in PrP, associated with familial prion disease in humans, in regions surrounding the GRR were also analyzed to determine their influence on prion infection. We found that these pathogenic mutations created protease sensitive PrP^Sc that was highly infectious when bioassayed in mice. Together these data show the high conservation and requirement of the residues of the GRR in prion infection gives it a potential role in targeted therapeutics for prion disease. Furthermore, this cell model also provides a unique model for testing the relationship between protease resistance and infectious forms of PrP which have been identified previously in animal models.

PPo6-22: Keep the Balance: The Pros and Cons of Cholesterol for PrP^Sc Propagation

Sabine Gilch,^1,2 Christian Bach,¹ Ina Vorberg¹ and Hermann M. Schätzl^1,2

¹Institute of Virology; Technische Universtität München; Munich, Germany; ²University of Wyoming; Depts. of Veterinary Science and of Molecular Biology; Laramie, WY USA

Key words: cholesterol, trafficking, rab proteins

Cellular cholesterol content is tightly regulated by endogenous synthesis and uptake from external sources. Inhibition of cholesterol synthesis or its extraction and complexation at the plasma membrane, respectively, can interfere with PrP^Sc propagation in prion-infected cultured cells and animals. Otherwise, we found that accumulation of cholesterol in late endosomes/lysosomes increases the degradation rate of PrP^Sc. Interestingly, cellular PrP^Sc content in such cells can be partially restored by overexpression of rab 9, which increases recycling of cholesterol and possibly of PrP^Sc to the trans-Golgi network. On the other hand, cellular cholesterol metabolism is modulated by prion infection. Highly controlled microarray studies revealed an upregulation of genes involved in cholesterol metabolism in prion-infected cell lines and primary neurons, which results in increased cholesterol levels. This can lead to perturbations in endosomal vesicle trafficking, and consistent with this we detected alterations in the membrane association of certain rab proteins in prion-infected cells. Surprisingly, rab 9 overexpression reduces PrP^Sc, indicating that v production is highly sensitive to alterations in dynamics of vesicle trafficking. We suggest that prions interfere with cholesterol metabolism and subsequently with vesicle trafficking in order to enable and sustain their propagation in cells.

PPo6-23: Cell-type and Prion Strain Independent Effects of Glycosaminoglycan Inhibitors

Hanna Wolf,¹ Romina Bester,¹ Ann-Katrin Felux,¹ Andrea Graßmann,¹ Kim Dietrich,¹ Hermann Schätzl^1,2 and Ina Vorberg³

¹Institute of Virology; Technische Universität München; Trogerstrasse, Munich Germany; ²Departments of Veterinary Science and of Molecular Biology; University of Wyoming; Laramie, Wyoming USA; ³Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE); Bonn, Germany

Prion diseases are associated with the conversion of a host encoded protein, PrPC, into an abnormal aggregated isoform, PrP^Sc, in the brain of the infected host. The precise mechanism of PrP^Sc formation and the cellular requirements for prion replication remain elusive. Co-factors implicated in prion biogenesis are glycosaminoglycans (GAGs), highly sulfated unbranched polysaccharides abundantly expressed on the cell surface. In murine cells persistently infected with prion strain RML sulfated GAGs have been shown to influence PrP^Sc internalization and PrP^Sc generation. However, so far it is unknown if these compounds also affect PrP^Sc formation and the establishment of persistent infections by different prion strains. We have studied the influence of GAG mimetic DS 500 and GAG desulfation by sodium chlorate on PrP^Sc formation and PrPC metabolism in neuronal and fibroblast cells. Both treatments greatly impaired persistent infections with two different prion strains independent of the cell type, suggesting that GAGs are generally required for efficient PrP^Sc accumulation. Since DS 500 increased cellular PrPC clearance while desulfation had the contrary effect, changes in total or cell surface PrPC levels unlikely account for the anti-prion effects of these compounds. Interestingly, during acute stages of infection, desulfation neither prevented PrP^Sc uptake nor the establishment of a prion infection. By contrast, PrP^Sc from the inoculum was taken up by the cells in the presence of DS 500, but unable to initiate an infection. Our results suggest that both compounds target different stages of prion infection.

PPo6-24: Divergent Effects of Caveolin-1 Knock-down on PrP^Sc Accumulation in de novo and Persistently Infected Cells

Andrea Graßmann,¹ Romina Bester,¹ Gloria Lutzny,² Hanna Wolf,¹ Kim Dietrich,¹ Hermann Schätzl^1,3 and Ina Vorberg⁴

¹Institute of Virology; Technische Universität München; Trogerstrasse, Munich, Germany; ²Third Medical Department; Technische Universität München; Klinikum rechts der Isar; Munich, Germany; ³Departments of Veterinary Science and of Molecular Biology; University of Wyoming; Laramie, Wyoming USA; ⁴Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE); Bonn, Germany

A conformational change of the glycosyl-phosphatidylinositol anchored cellular prion protein, PrPC, leads to the formation of disease-associated PrP^Sc, which likely constitutes the infectious agent causing prion diseases. The mechanism of prion entry and the initial stages of prion infection still remain enigmatic. In several cell types PrPC is associated with lipid rafts or caveolae, specialized raft subdomains enriched in caveolin-1 (cav-1). Rafts and caveolae likely also play a role in the internalization and formation of abnormal prion protein. We have investigated the role of cav-1 on de novo and established prion infections in mouse fibroblastoma cells. Here we show that in L929 cells PrPC localizes to detergent-resistent membrane fractions enriched in cav-1, confirming that also in fibroblasts PrPC is present in raft domains. Interestingly, siRNA-mediated knock-down of cav-1 did not impact de novo infection of cells with two different prion strains, suggesting that caveolae are neither essential for PrP^Sc uptake nor for the first stages of prion infection. However, in persistently infected cells, cav-1 knock-down significantly reduced PrP^Sc levels. Thus, caveolae/raft-dependent endocytosis is important for PrP^Sc accumulation in persistently infected fibroblast cells. If caveolae are directly involved in PrP^Sc formation or if cav-1 knock-down impairs trafficking of PrPC or of essential co-factors for PrP^Sc formation needs to be elucidated. Our results argue that cellular trafficking pathways necessary for PrP^Sc accumulation differ during acute and persistent prion infections.

PPo6-25: GPI Anchored and Anchorless PrP^Sc Isoforms in Prion Disorders

Michele Fiorini,¹ Kimberly Meade-White,² Jan PM. Langeveld,³ Cristina Casalone,⁴ Lorenzo Capucci,⁵ Bruce Cheesbro,² Salvatore Monaco¹ and Gianluigi Zanusso¹

¹University of Verona; Verona, Italy; ²Labratory of Persistent Viral Diseases Rocky Mountains Laboratories; Hamilton Montana, USA; ³CVI of WageningenUR; Lelystad, The Nederlands; ⁴IZSPLVA; Turin, Italy; ⁵IZSLER; Brescia, Italy

Key words: glycophosphatidyl-inositol anchor, two-dimensional analysis, prion protein, TSE

Introduction. Normal cellular and abnormal disease-associated forms of prion protein contain a C-terminal glycophosphatidyl-inositol (GPI) membrane anchor. The role of the GPI membrane anchor in prion diseases is unclear but many data suggest that it is involved in the abnormal prion protein formation and prion disease pathology. We performed a biochemical typing of GPI anchored and anchorless prion isoforms in animal and human prion disorders.

Results. 2D-PAGE analysis showed that PrPSc migration in GPI- mice samples was faster compared to wild-type mice, shifting of about 1 pH unit to the acidic pole. The contribution of the GPI anchor was the same before and after PK and PNGase treatment. We extend the biochemical analysis to all different sCJD molecular subtypes as well as to classical BSE and to both atypical H-type and L-type BSEs, showing the presence of both GPI+ and GPI- patterns.

Materials and Methods. Brain samples from RML GPI anchored (GPI+) and anchorless transgenic (GPI-) mice, from sCJD subtypes and classical and atypical BSEs were analyzed by SDS-PAGE and 2D-PAGE and immunoblot, before and after enzymatic PK digestion and PNGase F treatment.

Conclusion. In this study we defined the molecular coordinates of the GPI anchor, showing that these do not substantially differ in humans and animals, in normal and pathological conditions. We conclude that 2D-analysis represents a valid approach for detecting GPI+ and GPI- forms of PrPSc.

PPo6-26: PrP Knockout Zebrafish as a Novel Tool for in vivo Studies of PrP Function

Valerie C. Fleisch,^1,2 Gary Ritzel,² Laura Pillay,² Adina R. Bujold,^1,2 Andrew J. Waskiewicz² and W. Ted Allison^1-3

¹Centre for Prions & Protein Folding Disease; ²Department of Biological Sciences; ³Department of Medical Genetics; University of Alberta; Edmonton, Alberta USA

Key words: zebrafish, zinc finger nuclease

Introduction. Special interest in prnp knockout zebrafish derives from neurodevelopmental and early behavioural phenotypes we have observed during transient gene knockdown; this sharply contrasts a lack of developmental phenotypes in knockout mice. Targeted gene knockout is a rare feat in zebrafish, but the potency of this animal model for economical in vivo investigation has compelled us to refine and deploy the Zinc Finger Nuclease (ZFN) knockout technology (adapted from Meng et al. Nature Biotechnology 2008).

Results. Selected ZFN pairs showed specific cutting of prnp in an in vitro assay. Genomic mutations were identified in injected zebrafish embryos at a frequency of about 5%. Several carriers of this F0 generation gave rise to offspring mutated in prnp, confirming germ line transmission. F1 generations including heterozygous mutants have been raised and incrosses to generate prnp homozygous null fish are imminent.

Method. Our ZFNs are targeted to induce frameshifts in the 5' end of the prnp gene, producing null mutants. Custom ZFNs were created using two rounds of bacterial-one-hybrid selection to identify 5p and 3p zinc finger arrays binding to the chosen target site.

Conclusion. We have refined the challenging ZFN technology to create PrP knockout zebrafish. Considering previous knockdown data, this resource is expected to provide insights into PrP function during development and neuroprotection. This work also enables high-throughput in vivo screens of PrP molecular interactors and small molecule effectors of PrP function that will represent potential therapeutics.

PPo6-27: N-glycans and Sorting of the Prion Protein in MDCK Cells

Berta Puig,¹ Hermann Altmeppen,¹ Catharina Conrad² and Markus Glatzel¹

¹Institut of Neuropathology; Universitätsklinikum Hamburg-Eppendorf; Hamburg, Germany; ²Philipps-Univeristät Marburg; Marburg, Germany

Key words: prion protein, N-glycans, cell polarisation, MDCK cells

Until very recently it was believed that all glycosylphosphatidylinositol (GPI)-anchored proteins were sorted exclusively to the apical membrane in Madin-Darby canine kidney (MDCK) cells. This is not the case for the prion protein (PrP) which can mainly be found in the basolateral membrane in MDCK or Fischer Rat Thyroid (FRT) cells. N-glycans and the GPI anchor are sorting signals for GPI-anchored proteins such as PrP.

The aim of our study was to define the role of N-glycans on the targeting of PrP in MDCK cells. For this, we stably expressed 3F4-tagged PrP lacking the first, second or both glycosylation sites in MDCK cells. We also varied expression levels of the above mentioned glycomutants. By means of cell surface biotinylation assays and confocal immunofluorescence microscopy we investigated membrane localisation.

Here we show that all of the glycomutants are localized in detergent-resistant membranes (DRMs) and that the N-glycans do not play a major role in the basolateral sorting of PrP. Overexpression of PrP leads to missorting independently of glycosylation-status.

PPo6-28: Cytoplasmic PrP Dysregulates Signaling Pathways Regulating Protein Synthesis

Rory H. Shott,¹ Catherine Grenier,² Guillaume Tremblay,² Xavier Roucou² and Luis Schang¹

¹Department of Biochemistry and Centre for Prions and Protein Folding Diseases (CPPFD); University of Alberta; Canada; ²Department of Biochemistry; University of Sherbrooke, Canada

We developed, and tested on a simplified in vitro model, kinomic screens to identify signaling pathways critical in prion disease pathogenesis.

Abnormal cytoplasmic localization of PrP leads to cerebellar neuronal degeneration and gliosis. N2a cells expressing cytoplasmic PrP constitute simple models of such pathogenesis. CyPrPEGFP lacks ER-targeting and GPI-membrane-anchoring signals, leading to cytoplasmic PrP expression and protein synthesis inhibition. Our hypothesis is that the inhibition results from dysregulated signaling pathways.

Kinomic screens optimized for detection of 136 protein kinases in 1.2 mg protein were applied to N2a cells expressing CyPrPEGFP or, to allow clustering, CyPrPEGFP124stop and CyPrPEGFP157-231. Protein kinase levels were hierarchically clustered. Literature and database searches identified clusters containing kinases in given signaling pathways.

One cluster identified consists of kinases highly expressed in cells expressing CyPrPEGFP, and which participate in the CaMK4 pathway. Another consists of kinases with low expression, and which participate in the Akt/p70S6K pathway. Both pathways converge to regulate protein synthesis. Expression and activation of other kinases in, and phosphorylation of substrates of, these pathways confirmed dysregulation. Akt activates mTOR, which activates p70S6K to promote protein synthesis through eIF4B phosphorylation. Akt and mTOR were inhibited in cells expressing CyPrPEGFP. However, p70S6K activity and eIF4B phosphorylation were maintained. We are evaluating the MEK/Erk pathway, which also activates p70S6K.

Kinomic assays identified two dysregulated pathways. Akt and mTOR inhibition suggests a mechanism for protein synthesis inhibition. However, eIF4B phosphorylation was maintained. Cells expressing CyPrPEGFP may induce p70S6K via other pathways (e.g., MEK/Erk), attempting to maintain protein synthesis.

PPo6-29: Proteomic Analysis of N2a Neuroblastoma Cell Subclones

Minoru Tobiume,¹ Asuka Kurosawa,¹ Harutaka Katano,¹ Yoshio Yamakawa² and Tetsutaro Sata¹

¹Departments of Pathology; and ²Biochemistry and Cell Biology; National Institute of Infectious Diseases; Tokyo, Japan

Key words: N2a, 2D-DIGE

The conversion of the cellular prion protein (PrP^c) into a pathogenic isoform (PrP^Sc) is one of the underlying events in the pathogenesis of the fatal transmissible spongiform encephalopathies (TSEs). While the requirements of cellular co-factors have been suggested, the molecular and cellular mechanisms of the conversion remains poorly understand. The neuroblastoma-derived cell line N2a is one of the permissive cell lines for PrP^Sc infection. Several subclones derived from N2a were classified as PrP^Sc-susceptible or unsusceptible subclones (Uryu et. al. Microbi Immunol 2007; 51:661–9).

In this study, we compared the level of protein expressions in PrP^Sc-susceptible and insusceptible N2a subclones by using 2D fluorescence different gel electrophoresis (2D-DIGE) system. By image analysis, 25 spots were detected in the 2D gel. Mean intensities of these spots were varied two-fold between the two subclones. Using mass spectrometry (MS) analysis of selected protein spots, we identified ten different proteins, all of which proteins were upregulated in the PrP^Sc-susceptible subclone. Phenotypes of the selected proteins are currently under investigations.

PPo6-30: Complex Cellular Signals Triggered by PrPs During Zebrafish Development

Emily Sempou and Edward Málaga-Trillo

Department of Biology; University of Konstanz; Konstanz, Germany

Key words: PrP, zebrafish, development, cell-cell communication, signaling

PrP knockout mice have greatly contributed to our understanding of prion pathogenesis. However, the lack of clear phenotypes in these animals make it difficult to elucidate the physiological function of PrP and its connection to neurodegeneration. Recently, morpholino knockdown of PrP homologues in zebrafish (PrP-1 and PrP-2) revealed that these genes play important roles during gastrulation and neural development. The molecular basis of these defects is the ability of PrPs to engage in homophilic trans-interactions at cell-cell contacts, thereby triggering intracellular signals that further regulate cell-cell adhesion. Importantly, this role of PrP is shared by fish and mammals, making the zebrafish a ideal model to study conserved functions of PrP in cell-cell communication. Using genetic, cell biological and biochemical tools, we are currently investigating how PrP-1 regulates the stability of E-cadherin/ß-catenin adhesive complexes, the activation of Src tyrosine kinases and small GTPases, as well as the integrity of the actin cytoskeleton. Our results indicate that PrP-1 knockdown stongly affect the levels and subcellular localization of these molecules by influencing their proteasomal/lysosomal degradation and protein phosphorylation, respectively. Pharmacological and genetic rescue experiments support these observations. In addition, analysis of PrP-2 knockdown embryos suggests that these molecular networks are not only essential for zebrafish gastrulation, but also for neuronal differentiation and brain morphogenesis. Thus, analysis of PrP function in zebrafish embryos could help identify PrP functions related to the early onset of mammalian prion disease. This, in turn, may facilitate the search for novel therapeutic targets to block neurodegeneration.

PPo6-31: The Cellular Prion Protein Prevents Oxidative Stress-induced Autophagic Cell Death

Jae-Min Oh,¹ Richard I. Carp³ and Yong-Sun Kim^1,2

¹Ilsong Institute of Life Science and ²Department of Microbiology; College of Medicine; Hallym University; Anyang, Republic of Korea; ³New York State Institute for Basic Research in Developmental Disabilities; Staten Island, NY USA

Key words: prion, autophagy, oxidative stress

The cellular prion protein (PrPC) is involved in cell metabolism and maintenance such as copper metabolism and anti-oxidant activity. Autophagy can promote cell survival or cell death in response to various stresses and diseases. Our previous report showed that autophagy activity is increased in Prnp-deficient (Prnp^-/-) hippocampal neuronal cells compared to PrPC-expressing control cells under serum deprivation. Here, we investigated whether upregulated autophagy activity promote cell survival or cell death in Prnp^-/- cells under hydrogen peroxide (H₂O₂)-induced oxidative stress. We found that Prnp^-/- cells were more susceptible to oxidative stress in a dose- and time-dependent manner compared to control cells. In addition, markers for autophagy induction including the conversion from LC3-I into LC3-II by immunoblotting and LC3 punctate immunostaining were increased in H₂O₂-treated Prnp^-/- cells compared to control cells. Furthermore, we showed that blockage of autophagy with pharmacological autophagy inhibitor, 3-methyladenine, or shRNA knockdown of the autophagic gene, Beclin-1 or ATG5, significantly reduced the oxidative stress-induced cell death. Taken together, our study provides the first experimental evidence that PrPC is closely associated with oxidative stress-induced autophagic neuronal cell death.

Acknowledgements

This work was supported by a grant (Code #20080401034016) from BioGreen 21 Program, Rural Development Administration, and by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs (A085082), Republic of Korea.

PPo6-32: Normal Synovial Fibroblasts Express Both Prion and Shadoo. An Immunofluorscence Colocalization Studies

Martin Windsor and David Cullis-Hill

Sylvan Scientific; Sydney, Australia

Key words: prion protein, shadoo protein, synovial fibroblasts

Introduction. Synovial fibroblasts (SFs) form a monolayer of specialized cells that secrete synovial fluid into diarthrodial joints. It has been shown that fibrosarcoma and immortalized fetal fibroblasts can be infected with the scrapie agent. Shadoo (SPRN) is a recently identified member of the prion family which can form stable heterodimers with prion. The function of this complex has not been studied. Here we report on the expression of prion and shadoo in normal rabbit SFS.

Results. Clear distribution of both prion and shadoo (not doppel) was demonstrated. At the cell periphery prion and shadoo were mutually exclusive. There was clear colocalization of prion with shadoo in perinuclear cytoplasm. There was “on-on” colocalization of shadoo with cytoplasmic Z-DNA (a reverse helix form of DNA). Glypican-1 (a heparan-contain proteoglycan) and prion were also colocalized.

Methods. SFs were harvested from mature NZ white rabbits. Cultured SFs were fixed and stained with a panel of prion, shadoo and doppel anti-bodies in addition to nuclear and cytoplasmic markers.

Conclusion. Many studies of prion have employed abnormal cells (immortalized and/or neoplastic). SFs have a stable phenotype after serial subculturing and are easy to culture allowing all mammalian species to be examined. Their cytoplasm is spread and flat, allowing visualization of separate organelles for colocalization studies by using immunofluorescence. As a normal cell line they are a valuable model for invesigations of the functional interaction between shadoo and prion. Our data suggest a functional relationship between ZDNA, prion, shadoo and heparan sulfate proteoglycans.