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