Prion diseases - Basic Science

Dr. David WestawayCentre for Prions and Protein Folding Diseases,

University of Albertadavid.westaway@ualberta.ca

Spongiform change and amyloid in a scrapie-infected brain.

CWD in North America

BA

C D

The spectrum of prion diseases includes three patterns of manifestation

• Infectious (kuru, iatrogenic CJD, vCJDexperimental disease

• Familial (autosomal dominant genetic). F-CJD, GSS, FFI

• Sporadic (sporadic CJD, sporadic fatal insomnia).

How?

Are diseases like scrapie infectious or genetic?

HB “James” Parry, Univ Oxford: genetic, controlled by the recessive “s” gene

Alan Dickinson, Univ Edinburgh: naturally infectious

The search for a genomic nucleic acid

(Studies in the “slow virus” era)

Inactivation studies

• Ionizing radiation target size indicates an infectious particle of 55 kDa.

• UV irradiation indicates that if there is a double-stranded DNA genome it would have a size of about 40 base-pairs.

• Scrapie agent was resistant to agents that destroy or modify nucleic acids including psoralen, DNAses, RNAses, divalent cations.

PrPSc and PrPC

Gel of the purified prion protein (S=scrapie, N = normal

• A protease-resistant protein can be visualized in highly purified (~2000-5000 x) preparations of scrapieinfectivity made using detergent insolubility and sucrose gradient fractionations.

• This is called PrP27-30, because it has a size of 27-30 kDa.

• Although this species has implications for molecular nature ofprion infections, a slightly different type of experiment is done to use PrP27-30 as a diagnostic (below).

Harbingers of the “conformational hypothesis”

• PrPSc and PrPC have closely related amino acid sequence (1985).

• The PrP gene has a single uninterrupted coding exon (1986).• Low resolution structural analysis reveal PrPC is alpha-helical

(1992).• Low resolution structural analysis reveal PrPSc is enriched in β-

sheet (1992). Also, PrPSc amyloid deposits in hamster scrapiestain with congo red dye (1985).

• Overlapping Endo Lys C proteolytic fragments of PrPSc reveal no differences from the predicted sequence of PrPC (1993).

PrP encoding gene exon1 254

PrPC

PrP 27-30

PrPSc is host-encoded

Expression, maturation

Proteinase K digestion

PrPSc

Intronless gene DNA

S-S

CHO CHO

H1 H2 H3

Conversion

GPI

Cu domain

PrPC PrPSc

Structure

α-helix ~43% ~34%β-sheet ~3% ~43%Solubility + -Protease - +resistance + -

• Genetic and cell biological experiments are in favour of the prionhypothesis too.

PrPC PrPSc

α-helix ~43% ~34%β-sheet ~3% ~43%Solubility + -Protease - +Resistance + -Conformation is the key!

Financial Times: BSE

αCαB

CHOCHO GPI

PrP 27-30

(TM region)

β

90 232

142 amino acids, $6 billion : $42 million per residue

Some issues in prion replication.

• Does the PrPC get unfolded? (stable protein) and, if so, how?

• How does the unfolded protein (or native PrPC) get templated into a PrPSc-like shape?

PrPC PrP-U ? PrPSc

? ?

Heterodimer hypothesis (Prusiner)

• A large energy barrier prevents spontaneous conversion of PrPC to PrPSc.

• PrPC is unfolded by a hypothetical molecular chaperone called protein X.

• The replication intermediate is a PrPC /PrPSc

heterodimer (60 kDa).

PrPC PrP-U ? PrPSc

“Seeding” hypothesis (Caughey)

• There is only a small energy barrier between PrPC

and PrPSc but spontaneous conversion is prevented by a kinetic barrier: conversion is too slow.

• Once a pre-formed seed of PrPSc multimers is made the long lag period is avoided and PrPC to PrPSc

conversion takes place rapidly of the surface of the multimeric PrPSc.

• As multimers get bigger they fragment and thus can create multiple “new” seeds.

• Replication intermediates are big.

All labs agree “conversion”takes place on the cell-surface or in an early endosomal compartment

-distinction from viruses- (Implies an ectochaperone modulates folding)

Variant prion proteins modulate disease susceptibility

• Mouse PrP gene mutations• Sheep PrP gene mutations• Human PrP gene mutations

Methionine (M)129

Valine (V)129

M M M V V V

38% 51% 11%

Net genotype for polymorphism affects outcome of infectious, sporadic or familial prion diseases

Birefringent amyloid plaques in a prion disease (GSS)

Congo Red staining of Maltese-cross shaped GSS amyloid plaques.

Plaques can also be stained with thioflavin S, or with PrP-directed antibodies

Inheritance of a prion mutation tracks with disease in GSS families

Sporadic prion disease

• No families.• No clusters to indicate infectious spread• Disease “appears out of nowhere”• Presumably due to spontaneous misfolding of

PrPC.• Risk factors are unknown.

PrPC PrP-U ? PrPSc

Prion strains

• Strains are isolates of prions that can be distinguished in the same inbred host (typically mice or hamsters)

• Do not confuse with strains of mice !

Distinguishing prion strains (1)

• No nucleic acid to genotype

• Time-lapse to disease• Pattern of

neuropathology

Distinguishing prion strains (2)

• Sizes of protease-resistant PrPfragments on a blot

• Pattern of PrP glycosylation on a blot• Concentration of denaturant needed to

render the protein protease-sensitive

Strains are encrypted by different PrPSc conformations

Stable but distinct incubation time to disease onset, neuropathology, sizes of protease-resistant PrP fragments

Prion vs. viral infections

• Often (but not always!!!) have spongiform pathology post mortem.

• Sometimes have CNS amyloid, but there other CNS amyloidoses

• Will typically have protease-resistant PrP.• No genome.

Facts…

Prion vs. viral infections in practice

• If agent is DNAse-resistant does it automatically mean it is a prion infection?

• If agent is RNAse-resistant does it automatically mean it is a prion infection?

• If agent is protease-resistant does it automatically mean it is a prion infection?

• No genome, therefore no PCR assays.

• Absence of a nucleic acid is not a useful criterion for day-to-day diagnosis to distinguish from viral infections.

• But most types of prion infections will have protease-resistant PrP seen on a gel

(NB: need to use an antibody too for this western blot assay).

Prion Neuroinvasion

• Systemic route of PrPSc to the CNS, “centripetal spread”

Prion Neuroinvasion (contd)

• CNS to retina or muscle, “centrifugal”• Synapse “jumping” by PrPSc may

distinguish prion disease from non-transmissible amyloidoses– Cleavage adjacent to or within the

GPI anchor followed by receptor-mediated endocytosis?

– GPI anchor excision & insertion into membrane of the next cell

– Endocytosis and vesicle escape mediated by N-terminal PrP sequences

Neuronal “targeting”

• Spongy change• Synaptic deposition of PrPSc

• Extracellular plaques made of PrPSc

• Lysosomal perturbations

Different prion diseases affect different sub-cellular targets

? Different shapes of PrPSc have different trafficking and perturb different receptor mediated events ?

Part II: Prion protein physiology

• PrPC function• Doppel, the “dark shadow”• Shadoo, shadow of PrP

A: Neurophysiology of PrPC

• 17 PrP binding proteins identified to date.

• Signal transduction• Neuronal apoptosis• Binding to transition metals

and GAGs• Mainly on the cell-surface. • PrP k/o (Prnp0/0) mice have

two phenotypes.

(G.Millhauser)

Number of PrP copper sites at neutral pH

Substrate n Methodology Reference

PrP23-98 5.6 Equil. Dialysis (Brown et al, 1997)PrP53-89 4 CD (Viles et al, 2000)SHa23-98 5 ESI-MS (Whittal et al, 2000) PrP23-98 5 DEPC (Qin et al, 2002)PrP23-231 5 ESI-MS (Kramer et al, 2001)PrP23-231 4-5,5 DEPC (Qin et al, 2002)PrP121-231 4 EPR (Cereghetti et al, 2001)

CBA

CHOCHO GPI

+++++PrPC

1-4 5

Insert or ∆ , = F-CJD

The PrP-interactor proteome

Schmitt-Ulms et al, Nature Biotechnology 6:724-731 (2004)

Time-controlled transcardiac perfusion cross-linking: pull-down with α-PrP Fab HuM-D18

“Regular” PrP k/o mice

• Look pretty normal• Have normal CNS development, very

mild white matter changes in old age• Have disputed circadian rhythm deficits• Have disputed electrophysiological

deficits in the hippocampus• Have accentuated response to insults

Stroke models and hypoxia: PrPC, and PrP k/o mice

PrP staining near ischemic lesionsInfarct size, tetrazolium blue staining

“Irregular” PrP k/o mice

Prnp0/0 line

(wt)

Zrch I

NPU

Ngsk

Rcm0

ZrchII

Riken

Ataxia

No

No

No

Yes

Yes

Yes

YesActually activation of the next gene! - and blocked by reintroduction of PrPC

DplNeo

NMR structures of PrP and Doppel

(Huaping Mo,Jane Dyson)

Doppel protein made on developing sperm

Wild type

Knock-out

αCαBαA

CHOCHO GPI

PrPC

(TM region)Octarepeat

region

CHOCHO

αA αB/B’ αCGPI

Doppel

Brain + other tissues

Testis, PNS, adult cerebellum

CHO GPI

Areas of the Brain, retina

Shadoo

(HumanChr 20)

(HumanChr 20)

(Human Chr 10) (TM region)

Basic repeat region

Three members in the PrP supergene family, 2 expressed in the CNS

a)

b)

20

e10 e11 e12 e13 e14 e15 e16 e17

Sho

c)

Empty vector

Wild-type Sho

28

1714

Sho

*

Mouse ShadooGPI

Sho

PI-PLC (U)

d)

4938

28PrP

Sho22

16

36PNGaseF- -+ ++-

P1 P2 Adult

28

1714

Shoα-Sho(86-100)

α-rSho

α-PrP

f

a

α-Shadoopeptide04SH-1

j

e

k

d

ih

α-recomb.Shadoo06rSH-1

α-MoPrP7A12

α-HaPrP3F4

α-calbindin

cb

α-Shadoopeptide06SH-3

g

PatrickHorne

Interlocking expression of PrPC and Shadoo

Shadoo

CHOCHO

PrPOctarepeats

neuroprotectiveCHO GPI

αCαBαA

GPI

Random coil

• Need an unstructured domain: a site for protein/ protein interactions?

• Need a polyvalent binding site: scavenging or sensing?

• Action against different types of toxic stimuli

PrPC and shadoo trigger an ancient neuroprotective pathway