1-3-2011 Frank Baumann Prion Disease

8/6/2019 1-3-2011 Frank Baumann Prion Disease

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Prion Disease

Scrapie, Creutzfeld-Jakob Syndrome

Frank Baumann


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Literature

Aguzzi A, Sigurdson C, Heikenwaelder M (2008) Molecularmechanisms of prion pathogenesis. Annu Rev Pathol 3:11-40

Aguzzi A, Baumann F, Bremer J (2008) The Prions Elusive Reasonfor Being. Annu Rev Neurosci 31:439-477

Aguzzi A, Calella AM. (2009) Prions: Protein Aggregation and

Infectious Diseases. Physiol Rev 89:1105-1152


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Definitions of prions, PrPCand PrPSc

Prion (for proteinaceous, infectious only): Prions are the agents oftransmissible spongiform encephalopathy (TSE), with unconventionalproperties, such as resistance to high temperatures, relative high pressure,formaldehyde treatment or UV-irradiation.

The term prion does not have any structural implications other than that aprotein is an essential component of the transmissible agent.

PrPC

: The naturally occurring cellular prion protein derived from the prnpgene.PrPC in a given cell type is a necessary, but not sufficient for the replicationof prions.

PrPSc: An abnormal isoform of the mature prnp gene product found intissue of TSE sufferers. (1) Partially resistant to digestion by proteinase K,(2) believed to be conformationally distinct from PrPC and is considered tobe the transmissible agent.

These definitions were adapted from Prof. Adriano Aguzzi and Prof. Charles Weissmann.


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Conventional pathogens and infectious proteins

parasites

bacteria

fungi

viruses

Prions (-sheet rich proteins)


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Prion diseases: definiton of an infectious disease

Prion diseases are neurodegenerative diseases (spongiosis; gliosis;

PrP deposition) with invariably lethal outcome of the infected host.

Prion diseases are transmissible: An organ homogenate isolated

from an infected host (brain, secondary lymphoid tissues), injected

peripherally or intracerebrally into another host of the same species

or even a different species invariably causes the same disease

(species barrier).

The cellular prion protein is expressed on virtually every cell but to

high degree on cells of the CNS and on cells of the immune system.


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Kochs postulates on

proteinaceous agents1. The protein must be invariably present in a disease-

specific form and arrangement in the diseased tissue.

2. The physicochemical characteristics that conferinfectivity on a specific protein must be established.

3. The characteristics that render the host susceptible to

infection by a specific proteinaceous agent must beestablished.

4. The disease process must be induced in a susceptible

organism by the pure agent in its infectious form.5. The protein must be recovered in its infectious formfrom the animal that was experimentally infected withthe pure agent.


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PRIONS

Mink

Encephalopathy

Bovine Spongiform

Encephalopathy

(BSE)Feline

Encephalopathy

Chronic Wasting Disease

of Deer and Elk (CWD)Scrapie (sheep; goat)

ANIMALS

HUMANS

Creutzfeldt-Jakob

Disease(sCJD)

Kuru

vCJD;iCJD

Gerstmann-Strussler-

Scheinker Disease

Fatal Familial

Insomnia


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+NH3

The lipid anchored prion protein PrPC

Hydro

phobicity

amino acid number

mouse PrP

HC MASP H1 H3H2

repeats CD

-4

-3

-2

-1

0

1

2

3

4

1 2546432 23123 93 134 191

CC

GPI


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PrPC PrPSc-conversion

+NH3

adapted from Stan Prusiner


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Diagnostic Procedures for Prion Diseases


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The cellular prion protein PrPCand it`s pathogenic form PrPSc


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PrPCPrPSc

PrPSc is Proteinase K resistant

CJD AD CJD

+ Proteinase K


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Macroscopic view on Creutzfeldt-Jakob

CJD brain normal brain


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PrP deposition patterns in sCJD

plaque-like synaptic patchy-

perivacuolar


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Spongiosis Astrogliosis


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swollen

neuronal process

degeneratingorganelles

Courtesy of Prof. Steven DeArmond

Spongiform Encephalopathies:electron microscopical findings


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Human prion diseases

Caused by

peripheral uptake

of the agent

vCJD

iCJD

Kuru

Cause unknown sCJD

Caused bymutation in PRNP

fCJD

GSS

FFI

10-15%

80-85%sporadic

inherited

acquired

~ 160

> 400


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(Gambetti et al., British Medical Bulletin, 2003)

CJD types and subtypes

MM1 VV2 MV2 MM2 VV1 sFI MM2

2119

kDa

1. PrPSc-type in WB with unglycosylated peptides of 21 kDa

(type 1) and 19 kDa (type 2)

2. genotype at codon 129 (metionine/valine polymorphism)

(Parchi et al., Ann Neurol. 1999 Aug;46(2):224-33)

(Zanusso G et al., JBC, 2004)

1 2 1 2


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sporadic CJD variant CJD

Age of onset

Initial symptoms

PRNPGenotype(Codon 129)

PrPSc deposition

median 65 y median 29 (19-74) y

dementia,

visual disturbances

sensory, psychiatric

83% Met/Met 100% Met/Met

various deposition

patternsflorid

plaques

Distribution of PrPSc CNS CNS and lymphoid organs

Differences between sporadic and variant CJD


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vCJD

floridplaques

panencepahilcPrPSc deposition

type 4glycopattern

sCJD vCJD


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Template-directedrefolding

PrPC PrPSc

Very,very slow Rapid Rapid

Seeding(nucleation)

PrPC PrPSc

Variations of the prion hypothesis


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Aguzzi, A. et al. Physiol. Rev. 89: 1105-1152 2009;

doi:10.1152/physrev.00006.2009

Schematic representation of the protein misfoldingcyclic amplification (PMCA) reaction

S S


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Seeded PrPSc propagation and Formation of PrPSc

molecules de novo during serial PMCA propagation of

unseeded purified substrates.

.

Deleault N R et al. PNAS 2007;104:9741-9746


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PrPCPrPSc PrPSc

PrionStrain A

PrionStrain B

PrionStrain A

PrionStrain B

The strain phenomenon


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Very,very slow

Rapid

Offstate

Prionstate

Rapid

+

+

De novo priongeneration by

nucleation

Seed growth by recruitment ofmonomeric precursors

Prion replicationby fragmentation

Prion strain A

Strain B


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PrP immunoblots of prion-diseased human brain

Wadsworth J D F et al. PNAS 2008;105:3885-3890

2008 by National Academy of Sciences


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Adaptation of prion strains

elk,Hamster prions

C57BL/6 C57BL/6 C57BL/6600dpi 300dpi 150dpi

passage1 passage 2


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PrPSc deposition is strain specific

PK i t f P PSc i ifi f


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PK resistance of PrPSc is specific for

individual strains


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Pregnant wild-type mouse oroverexpressing prnp (tga20)

prnp tga20or wt

prnp0/0

Embryonic day 12,5

Is the endogenous PrPCneeded for toxicity:Grafting embryonic neural tissue into adult host mice

Brandner et al., Nature, 1996


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Development of pathological changes in the infected graft ?

Diffusion or active transport of PrPSc into the host brain ?

Reaction of the host brain ?

Infection of the CNS graft from extracerebral sites ?

End stage of the diseasein the graft ?

1

3

2

1

3

2

Goals of the Brain Grafting Experiments


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Grafts develop gliosis &spongiosis after i.c.inoculation

PrPSc plaques form in thehippocampus of the Prnpo/o

hosts.

Prnpo/o

hosts do not developclinical scrapie.

Sebastian Brandner et al.,Nature (1996) 379, 339-343

Lateral ventricle

Graft

Corpus callosum

Intracerebral Inoculation of PrP expressing Neurografts


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+NH3

The lipid anchored & the anchorless

prion protein PrPC

+NH3


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Pathology of inoculated anchorless PrP mice

Chesebro et al. 2005

T i i i t


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Transmission experiment

WT PrPSc

PrPs Tg mousePrPs Tg mouse

Plaque loadedclinically healthy

WT mouse

WT PrP

Sc

Time

Time

WT mouse

Plaque loaded dead

WT mouse

PrPSc

Time

WT mousePlaque loaded dead


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Acceleration of scrapie clinical disease in mice

expressing both anchorless PrP and wild-type PrP

Chesebro et al. Science 2005


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Results

Anchorless PrP can be converted into the

disease associated PrPSc It reduces the disease duration when co

expressed with membrane anchored PrP

C

However mice expressing only anchorless

PrP do not show clinical signs of scrapie,

while brain homogenates of these miceare infectious to PrPwt-mice

Th h i f


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Molecule

1

Molecule

2

Molecule3

CellX

CellY

CellZ

InfectiousPrions Central

nervous system

The mechanics of

neuroinvasion

Mouse models of peripheral prion pathogenesis:


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Mouse models of peripheral prion pathogenesis:Presence of prion infectivity over time

1week

4-5weeks

4months

6months

priontiter

C57BL/6 mice inoculated i.p.

with RML prion strain

Central

Nervous

System

Lymphoid organs(MLN, ILN, spleen)

200 dpi

6logLD50

240 dpi

3logLD50

8months

D t b l ll l i t i


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Prnp+/+

Footpad intraperitoneal

Prnpo/o

Lethal irradiation of prnp-/-recipient

Transfer of Prnp+/+fetal liver cells

Peripheral prion inoculation

Does extracerebral cellular prion protein

enable prion neuroinvasion ?

oral

intravenous


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Possible actors of Prion Neuroinvasion

FDCPrP

+

B

Prions

PrP+ or PrP-Lympho-

toxin-

M-cells?

Peyerspatch

Gastrointestinal tract

Sympatheticparasympathetic

FDC Macroph., DC (?)

Complement?

PrPB

PrPSc aggregationMicroglial activation

Synaptic damage

Neuronal apoptosisAmyloid deposition

Blood?

H ti d bl d i l d i


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Human tissues and blood involved in

propagation and transport of prions.


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A few Basic Questions to be addressed:

What is the physiological function of thenormal prion protein, PrPC?

The Problem:

Ablation of PrP in most mouse models does notinduce any severe pathological phenotype.

However, in some PrP-knockout strains animalsshow a neurological phenotype at an age of 200days or more.

Transgenic Proteins I


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Transgenic Proteins I

PrP: C:F: CD:


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Summary Survival curves PrPCD

Phenotype


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Phenotype


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Summary & Conclusions

Deletion of a specific internal domain of PrPinduces a fulminant, lethal phenotype with

extensive global axonmyelinic degeneration. Preferentially longest axons are affected.

This phenotype can be rescued dose-dependently by coexpression of wild-type PrPC.

Transgenic Proteins II


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Transgenic Proteins II

Dpl: CD_Dpl: PrP_Dpl:PrP:

Survival of transgene positive animals in


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0 200 400 600 800 10000

20

40

60

80

100

age [days]

survival[%

]

0 200 400 600 800 1000

0

20

40

60

80

100

age [days]

survival[%]

Dpl_CD+/- Prnp+/o

Dpl_CD+/- Prnpo/o

PrP_Dpl+/- Prnp+/o

PrP_Dpl+/-

Prnpo/o

MA

H

ydroph

obicity

HC

CD

CCSP

HC MASP

repeats CD

Hydrophobicity

CC

Survival of transgene positive animals in

the presence or absence of PrP


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Summary & Conclusions

Deletion of a specific internal domain of PrPinduces a fulminant, lethal phenotype with

extensive global axonmyelinic degeneration. Preferentially longest axons are affected.

This phenotype can be rescued dose-dependently by coexpression of wild-type PrPC.

Addition of this specific internal domain to a non

related but structurally similar scaffold detoxifiesthe phenotype of this otherwise lethal transgene

M d l f P P f ti


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Model of PrP function

Endocytosis via

clatrin coated pits

or caveolae

Interaction with

other TM-proteins in

cis

Interaction with

other TM-proteins in

trans

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1-3-2011 Frank Baumann Prion Disease