searching for custom dominant negative prps by in vitro ... 2010.pdf · isidro ferrer’sgroup ilia...
TRANSCRIPT
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Joaquín Castilla, PhDCJD 2010 and the CJD Foundation Family Conference
July 2010
The problem
Prion diseases are neurodegenerative disorders where a self-replication protein is responsible for the damage
The intrinsic mechanisims involved in the self-replicationrequirements are unknown
The data
Endogenous PrP is essential for the self-replication maintenance
There are purportely prion resistant species
PrPSc can be degradated if its replication is not maintained
The solution
Generation of KO animals
Mouse
Cow
Diminishing the expression level of PrP
Blocking the PrP replication
DrugDominant negative PrP
Dominant Negative PrPs
How to find them?
A PrP able to interfere the PrPSc replication by any mechanisims
Randomly
Observing the NatureFunction!!!!
Imitating the Nature (drugs)
The data
Purportely resistant species
Observation
•Experimental challenges
•Zoos experience
•No cases in Nature
The data
Purportely resistant species
Why?
Special PrPs unable to be converted due to the structural characteristics
Other factors or genetic background
Dominant Negative PrPsCorrelation between prion resistant species and prion resistant PrPs
We have
PrPs that are not very well converted
vs We need
PrPs able to interfere partiallythe replication of other prions
Dominant negative PrPs which couldcompromise the PrP functions
Too many nice in vitro studies
Many species resistants to manyprion strains
PrPs that are not converted at all
PrPs able to interfere completelythe replication of other prions
Dominant negative PrPs with thesame PrP functions
Nice in vivo studies
Resistant species to all prion strains
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Nature observation Selection of the right species
In vitro studiesComparative studies
In vitro studies
Selection of the right substituion
A long way
Structural analysis
In vivo studies
PROTEIN MISFOLDING CYCLIC AMPLIFICATION(PMCA)
PrPSc
PrPC
PrPPMCA
Incubation
Growing of units
Incubation
Growing of units
Incubation
Growing of units
NA A
Purportely resistant species
Prion strain Specie R1 R2 R3 R4 R5 R6 R7 R8
RML Mouse ND ND ND 100% 100% 100% 100% 100%
Me7 Mouse ND ND 100% 100% 100% 100% 100% 100%
22L Mouse ND ND 100% 100% 100% 100% 100% 100%
139A Mouse ND ND 25% 50% 100% 100% 100% 100%
79A Mouse ND ND 50% 75% 100% 100% 100% 100%
22F Mouse ND ND ND 100% 100% 100% 100% 100%
Scrapie Sheep ND ND 0% 0% 0% 0% 0% 0%
CWD Deer ND ND ND 50% 100% 100% 100% 100%
BSE Cow ND ND ND 0% ND ND 100% 100%
Unseeded None 0% 0% 0% 0% 0% 0% 0% 0%
Eight rounds of serial automated PMCA using rabbit brain as substrate
The values show the % of positive tubes out of the total number of tubes used (n=4 or n=6).
Control
-+ + + ++ +
NZW rabbit
Negative
PK -+ + + ++
Inocula
PK -+ +PK -+ + + +
No conversion
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Nature observation Selection of the right species
In vitro studiesComparative studies
In vitro studies
Selection of the right substituion
A long way
Structural analysis
In vivo studies
Comparative studies
Rabbit
Primary sequence
Horse
Cat
Dog
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Nature observation Selection of the right species
In vitro studiesComparative studies
In vitro studies
Selection of the right substituion
A long way
Structural analysis
In vivo studies
DOGSHEEPCOWRABBITCATELKPIGHUMANMOUSE
Prion protein NMR from different species
CAT HUMAN MOUSE
COW SHEEP DOG
Prion protein NMR from different species
Prion protein NMR from different species
CAT
DOG
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Nature observation Selection of the right species
In vitro studiesComparative studies
In vitro studies
Selection of the right substituion
A long way
Structural analysis
In vivo studies
90 2401
1: It appears also in susceptible species, like Armenian hamster, raccoon and red squirrel.
2: It appears also in bat, nilgai and anteater. The change does not imply a charge change (both are positive charged amino acids).
3: It is not described in other species. This change implies an important change of charge (from positive charged to negative charged)
2 3
CAT
DOG
Choosing the right substitution
Choosing the right substitutionInhibition studies
-C
+ + + +
10-6 10-5 10-4 10-3
+CAT
+ + + +
10-6 10-5 10-4 10-3
+RABBIT
+ + + +
10-6 10-5 10-4 10-3
+DOG
Standard brain-PMCA using mouse brain as substratie +different brain species
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Nature observation Selection of the right species
In vitro studiesComparative studies
In vitro studies
Selection of the right substituion
A long way
Structural analysis
In vivo studies
wt-PrP mut-PrP
pCMVTransient
transfectionpCMV
Transient transfection
KO cells KO cells
PCMA substrate
preparation
PMCA substrate
preparation
Similar PrP levels
Cell-PMCA
Evaluating the effect of amino acid substitutions by cell-PMCA
Evaluating the effect of amino acid substitutions by cell-PMCA
In vitro replication studies
In vitro dominant negative (inhibition) studies
NA A C
PK + + + + + + -+ +
wt M1 M2wt M1 M2 KOKO
Evaluating the effect of amino acid substitutions by cell-PMCA
In vitro replication studies
A C
PK + + + + -
Evaluating the effect of amino acid substitutions by cell-PMCA
In vitro dominant negative (inhibition) studies
wt M1 M2 KO
Brain PMCA using brain + cell substrates
Searching for Custom Dominant Negative PrPs by In Vitro Prion
Replication Studies
Nature observation Selection of the right species
In vitro studiesComparative studies
In vitro studies
Selection of the right substituion
A long way
Structural analysis
In vivo studies
In vivo studies
Generation of transgenic mice carrying the dominant negative PrP
The proof of concept
In vitro studies (PMCA)
In vivo studies (Mouse inoculations)
Improving the dominant negative PrP
Additive effect studies
Searching for new dominant negative PrPs
Generation of transgenic mice carrying the dominant negative PrP
In vitro studies
PK
Dil.
+ + + + -10-2 10-3 10-4 10-5
KO
C
-C
+ + + +
10-2 10-3 10-4 10-5
wt
+ + + +
10-2 10-3 10-4 10-5
wt+KO
+ + + +
10-2 10-3 10-4 10-5
wt+Tg*
+ + + +
10-2 10-3 10-4 10-5
Tg
-C
+ + + +
10-3 10-4 10-5 10-6
VM+wt
+
10-7
+ + + +
10-3 10-4 10-5 10-6
VM+Tg
+
10-7
+ + + +
10-3 10-4 10-5 10-6
VM+KO
+
10-7
Generation of transgenic mice carrying the dominant negative PrP
In vitro studies
Generation of transgenic mice carrying the dominant negative PrP
In vivo studies
Two different transgenic mouse lines are being inoculated with the most common prion species/strains
Experiments ongoing
Dominant Negative PrPsFuture
To styduy new NATURAL “resistant” PrPs
To study the additive effect by in vitro replication
KURUSheep and goat in vivo dataOther speciesNon mammalian species
To mimic the effect by using chemical drugs
To evaluate the best scenario using wt/tg mice (the proof of concept)
To study different protein delivery methods in cell cultures
ACKNOWLEDGEMENTS
Scripps-Florida
Natalia Fernández-BorgesJorge de CastroMaría MárquezUjas Parikn
Claudio Soto’s group
Juan María Torres’ group
Umberto Agrimi’s group
Charles Weissmann’s group
Corinne Lasmézas’ group
Jean E. Jewell’s group
Moredun
VLA
NPU
Adriano Aguzzi’s group
Glenn Telling’s group
Hubert Laude’s group
Prionics
Anthony Willianson’s group
Sue Priola’s group
Bayron Caughey’s group
Martí Pumarola’s group
Isidro Ferrer’s group
Ilia Baskakov’s group
Carsten Korth’s group
Jesús Requena’s group
Tomás Mayoral’s group
Neil Cashman’s group
Diego Rincón’s group
Diego Ruano’s group
CIC bioGUNE
Natalia Fernández-BorgesIker UriarteNagore SacristánAlberto MarinaSaioa RodríguezNahima Etxevarria
MICINN - Plan Nacional
Gobierno vasco
CJD Foundation