P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Cellular uptake and degradation of PrP-TSE; Relationship to Infectivity Hermann M. Schatzl University of Calgary 1 Key questions addressed: - Is it easy to infect (cultured) cells with prions? For which prions do we have cell culture models? What restricts infection of cells? Do cells have mechanisms to degrade/clear prions? Can cells adapt to or counter-act prion infection? - How does this all relate to in vivo infectivity and are our surrogate markers (e.g. PrPSc) reliable… 2 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Phases of a prion infection in vitro 1) Acute (transient..., abortive...): hours/days 2) Semi-acute (towards chronic...): weeks 3) Chronic (persistent...): months, years (rare!) Adaption processes involved: a) Prions to host cells: - e.g. optimal subcellular compartment of conversion - right converting unit/not too much stability... b) Host cells to prions: - e.g. regulation PrPc, no immediate apoptosis, modifier/susceptibility genes… Conversion of PrPc into PrPSc Profound changes biochemical properties PrPSc prion PrPc α-helical soluble PK sensitive no aggregation not infectious β-sheet insoluble PK resistant (rel.) aggregation infectious Main surrogate marker: PK resistance PK: - + - + PrPc PrPSc P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Cell biology of conversion PrPc into PrPSc 5 Much more complex: trafficking/recycling events Prions have to rely on cellular machineries... Gilch et al., 2008 Krammer et al., 2009 6 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Propagation of TSE agents (prions) in cell lines: what do we have and what not... * No reliable models for propagating BSE and CJD prions** ** (although huge attempts!) * *: primary/field isolates difficult **: e.g. Fukuoka strain Adapted from Grassmann et al., Viruses (2013) 7 Neuronal or brain-derived cell lines susceptible to TSE agents/prions Cell designation Tissue of origin or cell type Species of origin Prion strain N2a neuroblastoma cell line mouse GT1 hypothalamic cell line mouse SN56 cholinergic septal cell line mouse Chandler,RML, 139A, 22L, C506, Fukuoka-1, FU CJD Chandler,RML, 139A, 22L, kCJD, FU CJD , M1000 Chandler, ME7, 22L HpL3-4 mouse 22L mouse 22L SMB hippocampal PrP-deficient cell line, upon ectopic expression of mouse PrP brain derived PrP-deficient cell line, upon ectopic expression of mouse PrP prion-infected brain cell mouse Chandler, 139A, 22F, 79A CAD catecholaminergic cell line mouse MG20 microglial cell line overexpressing mouse PrP pheochromocytoma cell line tg20 mouse RML, 22L, 22F, 79A, 139A, ME7 Chandler, ME7, Obihiro, mouse-adapted BSE 139A, ME7 CF10 PC12 rat HaB brain-derived cell line hamster Sc237 SH-SY5Y neuroblastoma cell line human sCJD brain material MDB primary brain cells, SV40 transformed mule deer CWD Adapted from Grassmann et al., Viruses (2013) 8 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Non-neuronal cells susceptible to TSE agents/prions Cell designation Tissue of origin or cell type Species of origin Prion strain C2C12 skeletal myoblast cell line mouse 22L L929 fibroblast cell line mouse 22L, Chandler, RML, ME7 NIH/3T3 fibroblast cell line mouse 22L MSC-80 Schwann cell line mouse Chandler MovS Schwann cell like from dorsal root ganglia tgov mouse PG127, SSBP/1, scrapie field isolates moRK13 epithelial cell line expressing mouse PrPC rabbit voRK13 epithelial cell line expressing vole PrPC rabbit Fukuoka-1, 22L, Chandler, M1000, mouse sCJD vole BSE ovRK13/ RoV9 epithelial cell line expressing ovine PrPC rabbit PG127, LA404, SSBP/1, scrapie field isolates elkRK13 epithelial cell line expressing elk PrPC rabbit CWD Adapted from Grassmann et al., Viruses (2013) Neuronal and non-neuronal cells (manly murine)!!!9 Majority: non-susceptible cell lines Many “classical” cell lines: e.g. HEK, MDCK, MDBK, CHO No cell lines propagating efficiently human or bovine prions Have they been tested: extensively (scrapie, CJD/vCJD, BSE, CWD) Yet: - Not predictable!!! - Cross-species transmission in vitro possible (see reconstituted RK13 cells) P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Is there a prion-specific phenotype? (e.g. vacuolisation, apoptosis, cell death...) Usually none!!! (infected and uninfected cells cannot be differentiated) GT1 GT1 ScGT1 ScGT1 ScGT1-trk9 (Schätzl et al., 1997) 11 Uptake: cellular requirements? - PrPc: no; cells without PrPc can do as well - Specific receptor for prions: some candidates, no clear-cut evidence - Unspecific process: very likely, e.g. in context of phagocytosis, pinocytosis, exosomal uptake, microsomal uptake (release prions) - Can be blocked: energy needed… - Trafficking events needed (“endocytosis”) Cell type specific: yes, very likely Probably many cells can uptake ‘prions/PrPSc aggregates’, but without endogenous propagation of prions Release and entry of ‘aggregates’: a common and non-understood phenomenon in neurodegenerative disorders! 12 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Uptake: many cells can do... uptake of PrPSc is independent of PrPc expression... ...although no propagation 13 Uptake and initial degradation strain 22L in various cell lines (1-7 days); WB & IF insulinoma cells N2a J774 macrophage BV-2 (microglia) BMDM insulinoma cells Gilch et al., 2007 Aguib et al., 2008 6 M GndHCl, epitope retrieval PrPSc specific 14 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Kinetics of prion propagation (acute infection) Detection of newly formed PrPSc/prions Cells expressing 3F4 epitope tagged PrP 22L (5 h) mAb only detects newly formed PrPSc, not inocula PrPSc Incubation Immunoblot and IF/confocal microscopy, epitope tagging strategy 8h 48 h 26 h +PK/mAb 3F4 Vorberg et al., 2004 a,b GndHCl + 3F4 From transient to persistent infection: Most infections are ‘abortive’ (transient) Acute: Cell A: 4/4 Cell B: 3/4 Vorberg et al., JID and JBC 2004 Persistent: Cell B: 1/4 Cell A: 0/4 Bioassay: No infectivity in cells with transient PrPSc formation 16 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Two phases of prion infection Initial exposure leads to PrPSc formation - Can be initialized in prion „resistant“ cells - and in cells permissive to specific strain Very early events might happen in many cells Establishment of persistent prion infection - Drastic PrPSc increase 48-72 hrs post infection (IF) Suggests that during this phase specific events or co-factors lead to persistent infection What co-factors or events are this??? What restricts prion propagation in vitro? Important caveat: Cell division in dividing cells ‚dilutes out‘ prions... I) PrP/prion-related: - PrPc expression required: w/o PrPc no PrPSc - species barrier events (somewhat predictable) - strain barrier events (not predictable) II) Host cells-related: - modifiers, susceptibility loci??? - cellular mechanisms which allow continuous propagation - propagation outbalances degradation/clearance - adaption events (host to prions and vice versa) - recycling events, good release 18 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Prion species barriers and strain barriers Relative or absolute species barriers as seen in vivo also exist in vitro (with mod.) Prion strains show restricted host cell tropisms 19 Prion species barriers are mainly due to PrP amino acid sequence differences A single aa exchange can cause non-susceptible/non-convertible Mouse PrP, selected exchanges strain 22L Maas et al., 2007 There is significant variability between PrPs of different species aa alignment of 77 species Wopfner et al., 1999 20 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Host cell-related factors Prions must rely on certain host cell machineries No propagation in autophagy-deficient cells Cellular processes: - subcellular trafficking (organelles) -- cholesterol pathways -- QC/degradation pathways -- autophagy pathways - exosomal release - finding the right compartment of conversion... and many more... compartment of conversion: glyco-profiless differ.... acute persistent Vorberg et al., 2004 21 Cell-to-cell infection: media vs. cell-associated Schatzl et al., 1997 Both plays a role, depends on cell context 22 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Host cells can counter-act infection Insulinoma cells ‘spontaneously’ lost infection: Cured themselves by down-regulating PrPc not all cell types PrPc & PrPSc fast event (no) PrPc & no PrPSc Aguib al., 2008 23 Can we experimentally overcome these restrictions? only partly... 24 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD PrPC expression is a prerequisite for prion infection: Ensure expression of appropriate PrPc (same species) Use a PrPc-deficient cell line (no endogenous PrPc can interfere) (RK13; Vilette et al., 2001; GpL: Maas et al., 2007) To much PrPc may negatively Interfere… Use of retroviral/lentiviral systems for production of cells stably expressing tagged PrPs Allows selective follow-up of newly generated PrPSc/prions (mAb 3F4/L42) Maas et al., JBC 2007 25 Novel approaches A) Primary cells, neurospheres, neural stem cells… Good susceptibility, low titers (but don’t dilute out…) B) Organotypic cultures/slice cultures C) Scrapie-cell assay (SCA) (C. Weissmann): very sensitive diagnostic read-out (available for mouse and CWD prions) Primary neurons: 22L Primary neurons tg mouse: sCJD Hannaoui et al., 2013, 2014 Bach et al., 2009 26 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Phenotypic alterations PrPSc Falsig et al., 2008 Campeau et al., 2013 PNAS 2003 27 Correlation to in vivo infectivity Bioassay: inoculate mice with cell lysates: end-point, dilution assay: titer ScGT1 cell lysates, diluted 106-10 cell equivalents, 5 mice each, 3 experiments: 5.8 log ID50/107 cells Schatzl et al., 1997 What we see in immunoblot (PrPSc) translates well into bioassays Titers in cell lines < to << than prion amounts in a brain 28 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Correlation to in vivo infectivity Reduction of PrPSc translates well into reduction of specific prion infectivity... Anti-prion drug, 10 days treatment (22L-ScN2a cells), then without drug for 0-25 days WB for PrPSc (no signal) Bioassay into tga20 mice (i.c.) Mock-treated control: 106 cell equivalents 92 days p.i. 104 cell equivalents 120 days p.i. Drug-treated: 106 cell equivalents 178 dpi 1/5 mice 2 log difference = 32 days Yun et al., 2007 16 days = 1 log 86 days: 5.4 logs (=limit of detection) 29 Key questions addressed: - Is it easy to infect (cultured) cells with prions? For which prions do we have cell culture models? What restricts infection of cells? Do cells have mechanisms to degrade/clear prions? Can cells adapt to or counter-act prion infection? - How does this all relate to in vivo infectivity and are our surrogate markers (e.g. PrPSc) reliable… 30 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Answers: - No, very difficult Not for human, bovine prions, primary isolates… A variety of factors, centered around PrPc Yes, (very) effective ones To a certain extent PrPSc very good marker (sensitive enough): -- no great risk to miss infection below detection limit… -- (cross-)contamination events unlikely… 31 Take home messages - Cellular prion infection is a delicate issue - Main purpose: study cell biology, test anti-prion compounds; as a diagnostic tool is secondary - (Cross)-contamination (inadvertent) with significant amounts of prion infectivity very unlikely - Bovine cells/prions, human cells/prions: cannot be infected on purpose… - Species and strain barriers: second layer of protection… 32 P10 - Evolving Detection Methods for CJD and vCJD By: Hermann Schaetzl, MD Acknowledgements Department of Comparative Biology & Experimental Medicine , University of Calgary Collaborators UofC Former group members: Institute of Virology, Sabine Gilch Technical University of Munich Markus Czub Stefanie Czub Schatzl group Robin Yates Sabine Gilch group (2013 UofC) Ina Vorberg group (2010 DZNE Bonn) Frank Jirik Aru Narendran Joenel Alcantara Sampson Law (lab manager) Shweta Sah (lab tech) Sandi Nishikawa (lab tech) Yuzuru Taguchi M.D./Ph.D. Recent collaborators Shikha Jain Ph.D. M. Groschup/Riems J. Tatzelt/Munich University of Manel Ben Aissa, Ph.D. H. Kretzschmar/Munich Wyoming (2010-2013) Basant Abdulrahman, Ph.D. R. Lewis/Logan (Wyoming Endowed Excellene Chair) S. Lichtenthaler/Munich Shubha Jain, graduate student D. Motzkus/Gottingen Amalia Rose, co-op student (UBC) Departments of Veterinary Sciences M. Gasset/Madrid Department of Molecular Biology J. Greenlee/Ames Tazrina Alrazi, graduate student K. Doh-ura/Sendai E. Kremmer/Munich Erin Brown (lab tech, part time) M. Baier/Berlin Angelo Bianchi (animal tech) R. Wickner/Bethesda A. Burkle/Konstanz M. Klein/E. Flechsig/Wurzburg H. Rezaei/Jouy-en-Josas C. Lasmezas/Jupiter V. Erfle/Munich H. Wagner/Munich 34
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