Prion Diseases: toward further reduction of animal experimentation

Transcription

1 Prion Diseases: toward further reduction of animal experimentation Moudjou M, Chapuis J, Pierre S, Mekrouti M, Reine F, Herzog L, Laude. H, Vilette D, Rezaei H, Béringue V INRA, Université Paris-Saclay, VIM Unit, team MAP Jouy-en-Josas, France S.Halliez

2 Transmissible Spongiform Encephalopathies (TSE) Prion Diseases * Fatal Neurodegenerative diseases Distroy the central nervous system Induce locomotor, behavioral and sensory disorders * Long Incubation period and clinically silent 5 years in Cow, 2 years in Sheep, up to 40 years in human * Absence of immune response * Transmissibles * Nature of the causal agent : Non Conventionnal Transmissible Agent

3 Prion diseases affect Animals and Human Scrapie in sheep and goats (since 1730) Bovine Spongiform Encephalopathy: BSE (since 1985) Creutzfeldt-Jakob Diseases CJD ( ) sporadic, genetic, iatrogene, or contagious (variant CJD) Chronic Wasting Disease in Cervids (mainly USA, Canada and South Korea (since 1960) 1st cases of CWD in Europe, Norway 2016

4 Nature of the Infectious agent: The Prion Protein that exists in two forms, the normal and abnormal one

5 The normal cellular Prion Protein PrP C Biological Functions : Cellular signalisation Synaptic transmission Cell adhesion (zebrafish) Copper Homeostasis Sleep Cycle Oxydatif Stress (Neuroprtotector) Neuron myelinisation Glycanes N-ter But unnecessary Protein No major phenotype in PrP knock-out (PrP 0/0 ) animals Structure tertiaire Ancre GPI Bi-glycosylated Mono-glycosylated Non-glycosylated Electrophoretic Profile

6 Prion : The Protein Only Hypothesis (S. Prusiner 1982) Double-face Protein Fatal Neurodegenerative Protein misfolding disease PrP Sc Amyloid deposit Conversion Polymerisation Normal Prion Protein PrP C Pathological Prion protein PrP Sc Sc: Scrapie PrP Sc fibrills Spongiosis PrP Sc PrP C PrP Sc PrP C PrPres PrP Sc fibrills Proteinase K

7 Prion Strains: A hot question at the heart of TSE studies In the same host, several Prion strains propagate with distinct phenotypic traits Prion strains differ by their own biological and physicochemical properties Incubation Time Strain 1 Strain 2 Electrophoretical profile of PrPres Strain 1 Strain 2 Lymphotropism Strain 1 Strain 2 21K 19K Brain regional PrPSc deposit Lesion Profile Strain 1 Strain 2 Size of the PrP Sc Assemblies Strain 1 Souche Strain 1A Strain 1 Strain 2 Strain 2 Low size Large size The Phenotypic differences reflect diversity of PrPSc Conformations/Assemblies

8 How to study Prions in the Lab? Bioassay Infectivity test. The most sensisitive one. Strain diversity Physiopathology 1- Cell Culture models PrPc Non Infected wt Infected Tissues sampling Normal PrPc: green Nuclei: blue Abnormal PrPSc: green Nuclei: in blue Tissues homogenate preparation 2- Purified recombinant PrP: Biophysical and structural studies Tg mice: Hu, Bo, Ov, Ha, Mo PrPC inocoulation Healthy sample : animals survive Infected sample: animals dye with incubation time from 2 months to years 3- In vitro Prion Amplification: *PMCA: Protein Misfolding Cyclic Amplification *QuiC: Quacking Induced Conversion

9 PMCA: Protein Misfolding Cyclic Amplification (Adapted from Saborio Permanne et Soto 2001) PrP Sc Neoformed Multiplication PrP Sc + Incubation 30 min 1 cycle Sonication 30s Incubation Sonication PrP C Brain Lysate 1 Round de PMCA (24-48h)

10 Brain and Cell based miniaturized bead-pmca (mb-pmca) Sample to be sested (Inoculum) PrP Sc Incubation Sonication Produit PMCA 10 fold serial Dilutions PrPc Teflon beads (Baskakov Lab) Transgenic healthy mice Brain Lysate PrPc Main PMCA Improvements : Decrease of sample volume by 3 folds Adaptation to 96 well microplate format Use of cultured Cell lysate Cultured Cell Lysate Moudjou et al., Mbio 2013 Lacroux et al., Plos Pathogens 2014 Moudjou et al., Sci Rep 2016 Chapuis et al., Acta Neuropath Com 2016 Igel-Egalon et al., Plos Pathogens 2017

11 Rabbit Kidney epithalial cell line (RK13) transfected to express PrPc of different species Hum5 cells: Humain PrP C 20 µm Ham2 cells : Hamster PrPC P2FJ6 cells: Ovine PrP C 20 µm

12 Example of prion amplification using both Cell based- and Brain based mb-pmca Ov-Cell-PMCA Brain-PMCA Tg338 mice Ov-PrP Scrapie Prion Round of 48 Hours 17 Log 10 (dilution inoculum) U U Hum5 Cell PMCA + Brain PrP 0/0 Brain-PMCA Tg650 mice Hu-PrP Human Variant Creutzfeldt-Jakob Log 10 (inoculum dilution) Round of 48 Hours 1 Round of 48 Hours Moudjou et al., Mbio 2013 Moudjou et al., Sci Rep 2016

13 Prions Top: Small particles Example of mb-pmca impact on the reduction of animal bioassays Biochemical fractionation of brain Prion particles assemblies using Sedimentation Velocity Gradient technique Bottom: Large particles Density Gradient Top Small Particles Bottom Large particles Infectivity Titration : mice mice mice mice mice mice To titrate one fraction : 36 mice If titration of 1/2 samples : need 450 mice If titration of 1/3 samples : need 300 mice Tixador et al., Plos Pathogens 2010 Laferrière et al., Plos Pathogens 2013

14 The Brain mb-pmca will use only one mouse brain that replace mice that would be necessary for the Bioassay With Cell based-mb-pmca, 0 mouse brain is needed for Scrapie prion. At worst, half PrP 0/0 brain will be necessary for other prion strain (human, hamster) PMCA Data obtained in 48 hours instead of several months with the Bioassay

15 Brain-PMCA and Cell-based PMCA : Conclusions Highly efficient methods for fundamental and applied studies Applied to animal and human health *Anti-Prion drugs screening, *Validation of decontamination procedures, *Human Blood diagnosis of vcjd UK prevalence of vcjd 1/2000 UK (based on retrospective appendix analysis) Brain and Cell based mb-pmca constitute a method that will participate to further reduce and even replace animal use in some Prion disease studies

16 Pespectives Amplify with high efficiency other prion strains using only cultured cell lysate as substrate Extrapolate the mb-pmca procedure to the amplification of some misfolded proteins involved in other neurodegenerative diseases such as Alzheimer and Parkinson diseases that start to be considered as a : Prion-Like Diseases (Protein Misfolding Diseases) One Protein (peptide) State A State B Normal Abnormal, Misfolded

17 Human Rezaei Vincent Béringue Jérôme Chapuis Davy Martin Pierre Sibille Michel Dron Hubert Laude Joan Torrent S. Halliez Fabienne Reine Laetitia Hezog Christelle Jas Sandrine Truchet

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19 MM 2014

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21 BSE and variant CJD Crisis: Epidemiological aspects BSE Diatry primary Contamination Secondary Transmission 178 cases in UK 27 in France vcjd vcjd Global Distribution of Iatrogenic CJDs Dura matter graft: 228 Surgical Instruments : 4 Cornea transplants : 2 Growth Hormone : 226 Gonadotropines : 4 Blood transfusion: 4 (UK)

22 First case of Chronic Wasting Disease in Europe in a Norwegian free-ranging reindeer Sylvie L. Benestad, et al., Vet Res 2016 No deers = No Job for Christmas! *Highly transmissible disease for which the expansion could be incontrolable. *Zoonotic risk Active Surveillance

23 Example of scrapie prion amplification using both Cell based- and Brain based mb-pmca PMCA products titration in mice Ov-Cell-PMCA Tg338 Ov-mice Brain-PMCA 38 Scrapie Prion Log 10 (dilution inoculum) U U 1 Round of 48 Hours PMCA Amplicons (48h) are as infectious as the brain of infected mice at terminal stage (2 months) Moudjou et al., Mbio 2013 Moudjou et al., Sci Rep 2016

24 The normal cellular Prion Protein PrP C Biological Functions : Cellular signalisation Synaptic transmission Cell adhesion (zebrafish) Copper Homeostasis Sleep Cycle Oxydatif Stress (Neuroprtotector) Neuron myelinisation Glycanes N-ter But unnecessary Protein Structure tertiaire Ancre GPI Primairy Sequence Signal Peptide MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSVMSRP LIHFGNDYED RYYRENMYRY PNQVYYRPVD QYSNQNNFVH DCVNITVKQH TVTTTTKGEN FTETDIKIME RVVEQMCITQ YQRESQAYYQ RGASVILFSS PPVILLISFL IFLIVG Glycosylphosphatidylinositol GPI Bi-glycosylated Mono-glycosylated Non-glycosylated Electrophoretic Profile

25 Prion Stains: A hot question at the heart of TSE studies In the same host, several Prion strains propagate with distinct phenotypic traits Prion strains differ by their own biological and physicochemical properties Incubation Time Strain 1 Strain 2 Electrophoretical profile of PrPres Strain 1 Strain 2 Lymphotropism Strain 1 Strain 2 21K 19K Lesion Profile Size of the PrP Sc Assemblies Brain regional PrPSc deposit Strain 1 Strain 2 Strain 1 Souche Strain 1A Strain 1 Strain 2 Strain 2 Low size Large size The Phenotypic differences reflect diversity of PrPSc Conformations/Assemblies

26 Extent of PrP Sc structural landscape? Ovine PrP tg338 mice > 4 pass Ovine PrP Sc (VRQ) can store >18 stable and distinct SSD

27 Caractéristiques histopathologiques des Prions : La Triade de Hadlow * Mort des neurones ** Spongiose (Formation de «trous» dans le cerveau) *** Astrocytose et Gliose Cerveau infecté Cerveau sain Spongiose Activation de cellules gliales

28 % PrPres Temps d Incubation Dose Infectieuse Pour la souche de tremblante rapide, les assemblages de petite taille dans le cerveau sont les plus infectieux Activité de conversion par PMCA In vivo In vitro PrPres 1000x A 1 A 2 Fractions (Tixador et al., PLoS Path 2010) (Laferrière et al., PLoS Path 2013) et ont un pouvoir replicatif plus important in vitro (mb-pmca)

29 % PrP abnormal RES % ± PrP SEM Sc ± SEM Cinétique d accumulation de la PrP Sc et de l infectivité des Prions dans le cerveau de souris PrP Sc Infectivité du cerveau Signes cliniques Signes cliniques LAN21K Fast LAN19K BSEov Nor98 Clearance % Incubation time Inoculation V.B, Non publié Inoculation (Nakaoke et al., 2000)

30 Extent of PrP Sc structural landscape? Ovine PrP tg338 mice > 4 pass Ovine PrP Sc (VRQ) can store >18 stable and distinct SSD

31 Prion cross-species barrier is controlled by structural fit between PrP C and the infecting prion strain sporadic CJD Variant CJD Wild-type mice Human PrP transgenic mice Bank vole

32 Modèle de Conversion et de Propagation des Prions (nucléation / polymérisation) Noyaux infectant : quelques molécules de PrP Sc Conversion élongation PrP C Normale Fragmentation Propagation Adaptée de Philippe Tixador