Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies

Transcription

1 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies Joaquín Castilla, PhD CJD 2010 and the CJD Foundation Family Conference July 2010

2 The problem Prion diseases are neurodegenerative disorders where a self-replication protein is responsible for the damage The intrinsic mechanisims involved in the self-replication requirements are unknown

3 The data Endogenous PrP is essential for the self-replication maintenance There are purportely prion resistant species PrP Sc can be degradated if its replication is not maintained

4 The solution Generation of KO animals Mouse Cow Diminishing the expression level of PrP Blocking the PrP replication Drug Dominant negative PrP

5 Dominant Negative PrPs A PrP able to interfere the PrP Sc replication by any mechanisims How tofind them? Randomly Observing the Nature Function!!!! Imitating the Nature (drugs)

6 The data Purportely resistant species Observation Experimental challenges Zoos experience No cases in Nature

7 The data Purportely resistant species Why? Special PrPs unable to be converted due to the structural characteristics Other factors orgenetic background

8 Dominant Negative PrPs Correlation between prion resistant species and prion resistant PrPs We have vs We need PrPs that are not very well converted PrPs able to interfere partially the replication of other prions Dominant negative PrPs which could compromise the PrP functions Too many nice in vitro studies Many species resistants to many prion strains PrPs that are not converted at all PrPs able to interfere completely the replication of other prions Dominant negative PrPs with the same PrP functions Nice in vivo studies Resistant species to all prion strains

9 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies A long way Nature observation Selection of the right species Comparative studies In vitro studies Structural analysis Selection of the right substituion In vivo studies In vitro studies

10 PROTEIN MISFOLDING CYCLIC AMPLIFICATION (PMCA) Incubation Incubation Incubation Growing of units Growing of units Growing of units NA PrPSc PrPC PrPPMCA A

11 Purportely resistant species

12 Eight rounds of serial automated PMCA using rabbit brain as substrate 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% The values show the % of positive tubes out of the total number of tubes used (n=4 or n=6).

13 Control - NZW rabbit

14 Inocula PK PK PK Negative

15 No conversion

16 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies A long way Nature observation Selection of the right species Comparative studies In vitro studies Structural analysis Selection of the right substituion In vivo studies In vitro studies

17 Comparative studies Primary sequence Rabbit Horse Cat Dog

18 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies A long way Nature observation Selection of the right species Comparative studies In vitro studies Structural analysis Selection of the right substituion In vivo studies In vitro studies

19 Prion protein NMR from different species DOG SHEEP COW RABBIT CAT ELK PIG HUMAN MOUSE

20 Prion protein NMR from different species CAT HUMAN MOUSE COW SHEEP DOG

21 Prion protein NMR from different species CAT DOG

22 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies A long way Nature observation Selection of the right species Comparative studies In vitro studies Structural analysis Selection of the right substituion In vivo studies In vitro studies

23 Choosing the right substitution DOG CAT : 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)

24 Choosing the right substitution Inhibition studies +CAT +RABBIT +DOG C Standard brain-pmca using mouse brain as substratie +different brain species

25 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies A long way Nature observation Selection of the right species Comparative studies In vitro studies Structural analysis Selection of the right substituion In vivo studies In vitro studies

26 Evaluating the effect of amino acid substitutions by cell-pmca wt-prp mut-prp pcmv Transient transfection pcmv Transient transfection KO cells KO cells PCMA substrate preparation Similar PrP levels PMCA substrate preparation Cell-PMCA

27 Evaluating the effect of amino acid substitutions by cell-pmca In vitro replication studies In vitro dominant negative (inhibition) studies

28 Evaluating the effect of amino acid substitutions by cell-pmca In vitro replication studies NA A C PK wt M1 M2 KO wt M1 M2 KO

29 Evaluating the effect of amino acid substitutions by cell-pmca In vitro dominant negative (inhibition) studies A C PK wt M1 M2 KO Brain PMCA using brain + cell substrates

30 Searching for Custom Dominant Negative PrPs by In Vitro Prion Replication Studies A long way Nature observation Selection of the right species Comparative studies In vitro studies Structural analysis Selection of the right substituion In vivo studies In vitro studies

31 In vivo studies The proof of concept Generation of transgenic mice carrying the dominant negative PrP In vitro studies (PMCA) In vivo studies (Mouse inoculations) Improving the dominant negative PrP Searching fornew dominant negative PrPs Additive effect studies

32 Generation of transgenic mice carrying the dominant negative PrP In vitro studies KO wt wt+ko wt+tg* Tg Dil. PK C C -

33 Generation of transgenic mice carrying the dominant negative PrP In vitro studies VM+KO VM+Tg VM+wt C -

34 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

35 Dominant Negative PrPs Future To styduy new NATURAL resistant PrPs KURU Sheep and goat in vivo data Other species Non mammalian species To evaluate the best scenario using wt/tg mice (the proof of concept) To study different protein delivery methods in cell cultures To study the additive effect by in vitro replication To mimic the effect by using chemical drugs

36 ACKNOWLEDGEMENTS CIC biogune Natalia Fernández-Borges Iker Uriarte Nagore Sacristán Alberto Marina Saioa Rodríguez Nahima Etxevarria Scripps-Florida Natalia Fernández-Borges Jorge de Castro María Márquez Ujas Parikn MICINN - Plan Nacional Gobierno vasco CJD Foundation 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