PRIONS diseases

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PRIONS diseases

• How a simple protein can become an infectious
  agent?

Jean-Pierre LIAUTARD
ImmunoPrion
 Centre détudes dagents Pathogènes et
Biotechnologies pour la Santé 
MADCOW
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Human prions diseases

• Creutzfeldt-Jakob diseases (CJD)
• First human prion disease described (1929)
• Is mainly a sporadic disease
• Maladie de Gerstmann- Sträussler-Scheinker (GSS)
• Is a genetic disease
• Fatal Familial Insomnia (FFI)
• Is a genetic disease
• Kuru (New Guinea)
• Is an infectious disease
• New-varaint CJD (nvCJD)
• Is an infectious disease

Kuru in New Guinea
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Animals prions diseasesall are infectious
Scrapie
MadCow, BSE
Chronic-Wasting-Disease
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Epidémiology Most of the CJD are sporadic.
Sporadic this means that appears spontaneously
without cause
Exemple in France The number of cases is
proportional to the population
density. Incidence is 1/1,000,000 all over the
world
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Epidemiology Some CJD are genetic.
There are families with CJD (or CJD-like syndrom,
GSS,FFI), the genetic suceptibility correlates
with a point mutation in the gene of prion
protein. Penetrance is complete.
Mutation dans le gène PrP
Syndrome
P102L GSS P105L GSS A117V GSS Y145stop
GSS D178N CJD ou FFI V180I GSS F198S GSS
E200K CJD V210I CJD Q217R GSS Insertion
Octa-repetition CJD
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Epidémiology Prion diseases are transmissible
All the prion diseases are transmissible by brain
injection. C. Gajdusek received the nobel price
for the transmission of CJD to apes. Scrapie can
be transmitted to mice or hamsters
D. Carleton Gajdusek
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Epidemiology Summary

• All prion diseases are transmissible, this means
  infectious
• Some prion diseases are sporadic, they appear
  spontaneously, without cause
• Some prion diseases are genetic.
• All hypothesis on the etiology should take into
  account this three specificities

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Prion diseases Histopathology

• There are two main characteristics
• Spongiosis
• Accumulation of a protein in the amyloid form

Prion protein
Amyloid of prion protein
Spongiosis
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What is the nature of the infectious agent prion?
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Discovery Purification of the infectious agent
In 1983 S.B. Prusiner found that the infectious
agent is a protein. He coined the term prion.
SB Prusiner
Affinity chromatography
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Surprise The  infectious protein  is coded by
the host genome
This protein, cloned by C. Locht, is expressed in
many tissues.
C. Locht
Exemple in human blood cells
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Structure the prion protein struture is common
STRUCTURE K. Wüthrich determined the structure
of the cellular prion protein. As a new surprise
the structure appears common, with no specificity
that could explain the behavior as an infectious
agent.
Kurt Wüthrich
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Remark Infection is associated with prion
proteinbut precipitated under amyloid
conformation
The disease correlates with the amyloid
formation. Amyloids are organized aggregates,
one-dimension crystal
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Confirmation The cellular prion protein is
necessary to sustain infection
Ch. Weissmann showed that prion-KO mice cannot be
infected
Ch. Weissmann
100
prp/
CD1
Scrapie symptom-free ()
50
prp/
prp/
0
100
300
200
400
500
Time after inoculation in days
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The prion conjecture
SB Prusiner
A misfolded prion protein would be the infectious
agent.
Question what is the molecular mechanism
involved?
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The prion conjecture Amyloid formation hypothesis
P. Lansbury proposed that amyloid formation
could explain prion propagation.
P. Lansbury Jr.
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The prion conjecture the amyloid hypothesis
What happens?
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Experimental results that sustain the amyloid
hypothesis
Infectious agent can be amplified in vitro
C. Soto
Principe of PMCA Protein Misfolding Cyclic
Amplification
Reproduit les caractéristiques de souches
Cell-free propagation of prion strains Joaquin
Castilla, Rodrigo Morales, Paula Saa, Marcelo
Barria, Pierluigi Gambetti and Claudio Soto
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Experimental results that sustain the amyloid
hypothesis
C. Soto
The amplified product is infectious
10-55
dilution
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Experimental results that sustain the amyloid
hypothesis
Amyloids prepared with recombinant prion protein
have been found infectious in two published
experiments
SB Prusiner
Nevertheless, this experiment succeeded only with
Tg99 transgenic mice that spontaneouly (genetic)
become illed.
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A simple quantitative model deduced from this
conjecture seems to explain all but!
Tnlag
Nucléation
Polymérisation
Tplag
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This model explains

• Tlag explains sporadic diseases
• Seeding explains infectious transmission
• Propensity to form amyloid the genetic disease
• Last but not least experimental results!

Expériences
Théorie
BUT!
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But!

• The results remain very qualitative
• In vivo results are not precise enough
• Too much parameters should be ad hoc estimated
• Many different models can also explain the
  observed results
• For instance

Be careful of the Canada Dry syndrome
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Furthermore some results are not explained!

• Most of the prion amyloids produced in vitro are
  not infectious
• Most of amyloidosis (diseases with amyloids
  Alzheimer, Parkinson, Diabetes,) are not
  infectious.

WHY???
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How to solve these contradictions?
Proposition
Accept the conjecture and design an experimental
approach that is precise enough to falsify a
quantitative model!
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To accept the conjecture, this means that in
vitro polymerization explain the totality of the
prion phenomenon
But, it is necessary to add new hypotheses to
explain why only some amyloids are infectious.

• The mechanism of prion polymerization is very
  specific
• Analyze the polymerization mechanism
• Find this specificity in the polymerization
  dynamic
• Only some specific structures are infectious
• Analyze the structure of amyloids
• Find the infectious structures

To be investigated experimentally
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Design of the experimental model
And Control of the results obtained
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Experimental model in vitro polymerization
Experimental
/- Amyloïde
rPrP
polymerization
Tht
hn
Fluo
Time (hours)
Incubation
We used ThT fluorescence that is proportional to
amyloid concentration.
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Control Amyloid structure of the fiber
Birefringence observed by Congo Red microscopy
Amyloid
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Control Amyloid structure of the fiber
Neg
Pos
2-3 nm fibres 5 nm spacing
Electron Microscopy (Negative staining)
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Control Amyloid structure of the fiber
Electron Microscopy (cryoanalysis)
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Measurment of the fibers length
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Control Amyloid structure of the fiber
1653 a
TFIR Spectroscopy
1626 b
Cross-Beta Amyloid specific
Amyloïde B Repris dans H2O
Amyloïde B Tp Phosphate Na pH 7.0
rPrP precipité
1543
1537
rPrP soluble
1516
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What are the advantages of the in vitro
polymerization studies?
Many quantitative parameters can be controlled or
measured

• Can be measured during experiment
• The total quantity of amyloid
• The length of the amyloid fibers
• The quantity of remaining protein (PrPC)
• The spatial structure (low resolution)
• Can be controlled during experiment
• Temperature
• Initial concentration
• Solution denaturant
• Agitation
• Can be deduced from the experiment
• Polymérization rate (1/t)
• Latency Time(Tlag)
• Mid-Time (T0)

Parameters deduced from experiments
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What are the advantages of the in vitro
polymerization studies?
Some parameters can be obtained by two
independent methods
SPR (Biacore) measurement of kinetic contants
Parameters deduced from experiments
Polymerization constants direct measurement (SPR)
Polymerization kinetics
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Some experimental results
That would necessary to take into account
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Spontaneous polymerization (Sporadic CJD)

• Tlag depend on the concentration while
• apparent rate of polymerization (Tau) no.
• Interpretation
• Tau depends on the structure of the amyloid.
• The dependence of the Tlag is in agreement
• with the nucleation process of the amyloid
• formation
• To be noticed
• In vivo, the increase of the concentration of
• the prion protein reduce the incubation time

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Seeded polymerization (infection)
Conclusions 1. The polymerization rate
increases at each passage. 2. Tlag is reduced at
each passage but does tend toward zero as
expected. This means 1. The system selects the
most rapid polymerizing structures. 2. The
system needs conformational changes to be able to
polymerize.
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Delayed seeding experiment
080312 ex-56
080312 ex-56
Tau
Tlag
1,2
7
1,0
6
0,8
5
0,6
4
Y Data
Heures
0,4
3
0,2
2
0,0
1
Non Ense
T3H
T2H
T1H
T0H
Non Ense
T0H
T1H
T2H
T3H

• The polymeiztion rate does not change
• TLag is reduced, but does not tend toward zero

Conclusion The amyloid formation needs
conformational changes both of the amyloid and
of the soluble prion protein
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Direct measurement of the polymerization rateSPR
analysis (BiaCore)

• Analysis
• No saturation
• Incomplete dissociation

This means

• Continuous polymerization
• There are two conformations
• One with rapid dissociation
• One with very slow dissociation

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Direct measurement of the polymerization rate
SPR analyzis (BiaCore) dissociation
Effectivelly, the analysis shows that two
dissociation rates are involved
k-2 10-3 s-1 k-1 10-2 s-1 And perhaps a
third k-3 10-5
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Designing Modelsadapted to mathematical treatment
That takes into account the experimental results
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Plymerization mechanismModel proposed The
 Dock-Lock  mechanism
Nouvelle étape
k1
k2
k-2
k-1
PM
PM
M
P

Transconformation (Lock)
Interaction (Dock)
Simulation Mathematica
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A general mechanism for polymerization
A chaperonning model
?
?
?
?
?
q
?
?
?
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Simulation (Runge-Kutta avec Mathematica)
Problem This look like experimental
results. Warning The Canada dry
syndrome! Solution To work with a
mathematician for modeling
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Team collaborations

• Prion Montpellier
• Alvarez-Martinez T.
• Arnaud J.-D.
• Fontès P.
• Zomosa-Signoret V.
• Mathematics Lyon
• Pujo-Menjouet L.
• IFR122
• Guerin M.C.

• INSERM Montpellier
• Torrent J.
• Amsterdam
• Peters P.

ImmunoPrion
MADCOW
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A mathematician is coming!