Prion Diseases: toward furtherreduction 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 MAP2
78350 Jouy-en-Josas, France
S.Halliez
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
Prion diseases affect Animals and Human
• Scrapie in sheep and goats
(since 1730)
• Bovine Spongiform Encephalopathy: BSE
(since 1985)
•Creutzfeldt-Jakob Diseases CJD (1920-1921)
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
Nature of the Infectious agent: The Prion Protein
that exists in two forms,the normal and abnormal one
The normal cellular Prion Protein PrPC
Biological Functions :✓Cellular signalisation✓ Synaptic transmission✓Cell adhesion (zebrafish)✓Copper Homeostasis✓ Sleep Cycle✓Oxydatif Stress (Neuroprtotector) ✓Neuron myelinisation
But unnecessary ProteinNo major phenotype in PrP knock-out
(PrP0/0) animals
Structure tertiaire
Glycanes
Ancre GPI
N-ter
Electrophoretic Profile
Bi-glycosylated
Mono-glycosylated
Non-glycosylated
Prion : The Protein Only Hypothesis (S. Prusiner 1982)
Double-face Protein
Conversion
PrPSc Amyloid deposit
Pathological Prion protein
PrPSc Sc: Scrapie
Normal Prion Protein
PrPC
➢ Fatal Neurodegenerative Protein misfolding disease
PrPSc fibrills
PrPC
PrPSc
PrPSc fibrills
PrPC PrPSc
- + - +
PrPres
Proteinase K
Spongiosis
Polymerisation
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
The Phenotypic differences reflect diversity of PrPSc Conformations/Assemblies
Lesion Profile
Lymphotropism
Size of the PrPSc Assemblies
Incubation Time
Strain 1 Strain 2
Electrophoretical profile of PrPres
Brain regional PrPSc depositStrain ASouche 1
Strain 1 Strain 2
Strain 1 Strain 2
Strain 1 Strain 2
Strain 1Strain 2
Strain 1
Strain 2
Low size Large size
19K21K
Bioassay Infectivity test. The most sensisitiveone.
Strain diversity – Physiopathology…
1- Cell Culture models
Infected PrPc wt Non Infected
Abnormal PrPSc: green
Nuclei: in blue
Normal PrPc: green
Nuclei: blue
Tissues sampling
Tissues homogenate
preparation
Tg mice: Hu, Bo, Ov, Ha, Mo PrPC
Healthy sample :
animals surviveInfected sample: animals dye
with incubation time from
2 months to years
How to study Prions in the Lab ?
3- In vitro Prion Amplification: *PMCA: Protein Misfolding Cyclic Amplification
*QuiC: Quacking Induced Conversion
2- Purified recombinant PrP: Biophysical and structural studies
inocoulation
PrPC
Brain Lysate
+
PrPSc
PMCA: Protein Misfolding Cyclic Amplification (Adapted from Saborio Permanne et Soto 2001)
1 Round de PMCA (24-48h)
Incubation
30 min
PrPSc Neoformed
Incubation
Multiplication
Sonication
Sonication 30s
1 cycle
10 fold serial
Dilutions
Sample to be sested(Inoculum)
PrPSc
Brain Lysate
Transgenic
healthy mice
PrPc
Brain and Cell based miniaturized bead-PMCA (mb-PMCA)
Incubation Sonication
Produit PMCA
Main PMCA Improvements : ❖ Decrease of sample volume by 3 folds
❖ Adaptation to 96 well microplate format
❖ Use of cultured Cell lysate
Teflon beads(Baskakov Lab)
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
…
❖ Cultured Cell Lysate
PrPc
20 µm
20 µm
Rabbit Kidney epithalial cell line (RK13)
transfected to express PrPc of different species
Hum5 cells: Humain PrPC Ham2 cells : Hamster PrPC
P2FJ6 cells: Ovine PrPC
Example of prion amplification using both Cell
based- and Brain based mb-PMCA
-9 -10 -11 -12 -13 U
38
28
17
Brain-PMCA
Tg338 mice Ov-PrP
Log10
(dilution inoculum)
Ov-Cell-PMCA
-8 -9 -10 -11 -12 U
Scrapie Prion
Moudjou et al., Mbio 2013
Moudjou et al., Sci Rep 2016
1 Round of 48 Hours
Human Variant
Creutzfeldt-Jakob
-5 -6 -7 -8 -8 -9 -10
Hum5 Cell PMCA+ Brain PrP0/0
Brain-PMCA
Tg650 mice Hu-PrP
38
28
17
Log10
(inoculum dilution)
1 Round of 48 Hours
1 Round of 48 Hours
Infectivity
Titration :10-1……... 6 mice
10-2……... 6 mice
10-3 ……...6 mice
10-4 …….. 6 mice
10-5 …….. 6 mice
10-6 …….. 6 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
Example of mb-PMCA impact on the reduction
of animal bioassaysPrions
Density
Gra
die
nt
Top:
Small particles
Bottom:
Large particles Top
Small ParticlesBottom
Large particles
17 18
Biochemical fractionation of brain Prion particles assemblies
using Sedimentation Velocity Gradient technique
Tixador et al., Plos Pathogens 2010
Laferrière et al., Plos Pathogens 2013
The Brain mb-PMCA will use only one mouse brainthat replace 300-400 mice that would be
necessary for the Bioassay
PMCA Data obtained in 48 hours instead of several months with the Bioassay
With Cell based-mb-PMCA, 0 mouse brain isneeded for Scrapie prion.
At worst, half PrP0/0 brain will be necessaryfor other prion strain (human, hamster)
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
❖ 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 :
Pespectives
State A State B
Normal Abnormal,
Misfolded
One Protein (peptide)
Prion-Like Diseases(Protein Misfolding Diseases)
Fabienne Reine Laetitia Hezog Christelle Jas Sandrine Truchet
Hubert
Laude
Human
Rezaei
Vincent
Béringue
Michel
Dron
Davy
Martin
Joan
TorrentPierre
Sibille
Jérôme
ChapuisS. Halliez
MM 2014
BSE and variant CJD Crisis: Epidemiological aspects
Diatry primary
Contamination
Global Distribution of Iatrogenic CJDs
Dura matter graft: 228Surgical Instruments : 4
Cornea transplants : 2
Growth Hormone : 226Gonadotropines : 4
Blood transfusion: 4 (UK)
Secondary
Transmission
vCJD vCJD
BSE
178 cases in UK
27 in France
First case of Chronic Wasting Disease in Europe
in a Norwegian free-ranging reindeer
Sylvie L. Benestad, et al., Vet Res 2016
*Highly transmissible disease for which the expansion could be incontrolable. *Zoonotic risk
Active Surveillance
No deers = No Job for Christmas … !
Example of scrapie prion amplification using both
Cell based- and Brain based mb-PMCA
-9 -10 -11 -12 -13 U
38
28
17
Tg338 Ov-mice
Brain-PMCA
Log10
(dilution inoculum)
Ov-Cell-PMCA
-8 -9 -10 -11 -12 U
Scrapie Prion
PMCA Amplicons (48h) are as
infectious as the brain of infected
mice at terminal stage (2 months)
PMCA products titration in mice
Moudjou et al., Mbio 2013
Moudjou et al., Sci Rep 2016
1 Round of 48 Hours
The normal cellular Prion Protein PrPC
Primairy Sequence
Biological Functions :✓Cellular signalisation✓ Synaptic transmission✓Cell adhesion (zebrafish)✓Copper Homeostasis✓ Sleep Cycle✓Oxydatif Stress (Neuroprtotector) ✓Neuron myelinisation
But unnecessary Protein
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
Signal Peptide
1
51
101
151
201
251
Glycosylphosphatidylinositol GPI
Structure tertiaire
Glycanes
Ancre GPI
N-ter
Electrophoretic Profile
Bi-glycosylated
Mono-glycosylated
Non-glycosylated
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
The Phenotypic differences reflect diversity of PrPSc Conformations/Assemblies
Lesion Profile
Lymphotropism
Size of the PrPSc Assemblies
Incubation Time
Strain 1 Strain 2
Electrophoretical profile of PrPres
Brain regional PrPSc depositStrain ASouche 1
Strain 1 Strain 2
Strain 1 Strain 2
Strain 1 Strain 2
Strain 1
Strain 2Strain 1
Strain 2
Low size Large size
19K21K
Extent of PrPSc structural landscape?
> 4 pass
Ovine PrP tg338 mice
➢ Ovine PrPSc (VRQ) can store >18 stable and distinct SSD
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
Pour la souche de tremblante rapide, les assemblages de petite taille
dans le cerveau sont les plus infectieux…
… et ont un pouvoir replicatif plus important in vitro (mb-PMCA)
Activité de conversion par PMCA
1000x
A1 A2
In vitro
(Laferrière et al., PLoS Path 2013)
(Tixador et al., PLoS Path 2010)
Tem
ps
d’I
nc
ub
ati
on
Do
se I
nfe
cti
eu
se
% P
rPre
s
Fractions
In vivo
PrPres
0 10 20 30 40 50 60 70 80 90 100
0
20
40
60
80
100
PrP
RE
S %
± S
EM
% Incubation time
LAN21K Fast
LAN19K
BSEov
Nor98
Cinétique d’accumulation de la PrPSc et de l’infectivité des Prions dans le
cerveau de souris
PrPSc Infectivité du cerveau
V.B, Non publié (Nakaoke et al., 2000)
Signes cliniques
Inoculation Inoculation
% a
bn
orm
al P
rPSc
±SE
M
Clearance
Signes cliniques
Extent of PrPSc structural landscape?
> 4 pass
Ovine PrP tg338 mice
➢ Ovine PrPSc (VRQ) can store >18 stable and distinct SSD
Wild-type mice
Bank vole
Human PrP transgenic mice
sporadic
CJD
Variant
CJD
Prion cross-species barrier is controlled by structural fit between PrPC and the
infecting prion strain
PrPC
Normale
Noyaux infectant :
quelques molécules
de PrPSc
Modèle de Conversion et de Propagation des Prions (nucléation / polymérisation)
Conversion
Propagation
élongation
Fragmentation
Adaptée de Philippe Tixador
