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