David Westaway
Title goes here
Prion diseases - Basic Science
Dr. David WestawayCentre for Prionsand Protein Folding Diseases,
University of [email protected]
Spongiform change and amyloid in a scrapie-infected brain.
CWD in North America
BA
C D
David Westaway
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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 scrapieinfectious 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)
David Westaway
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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
David Westaway
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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 of prion infections, a slightly different type of experiment is done to use PrP27-30 as a diagnostic (below).
David Westaway
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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.
David Westaway
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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
David Westaway
Title goes here
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. Identity of protein X is unknown
• The replication intermediate is a PrPC /PrPSc
heterodimer (60 kDa).
PrPC PrP-U ? PrPSc
Protein X might act here to unfold PrPC
“Seeding Hypothesis”, seeded growth of monomeric subunits into a multimer
David Westaway
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multimer
Remove, dilute and re-seed
“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.
David Westaway
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All labs do agree that “conversion”takes place on the cell-surface or in an early endosomalcompartment
-distinction from viruses- Implies a molecular chaperone outside the cell might modulate re-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
David Westaway
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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
David Westaway
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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
David Westaway
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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
David Westaway
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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 PrPsequences
David Westaway
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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)
David Westaway
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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 transcardiacperfusion cross-linking: pull-down with α-PrP FabHuM-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 PrPk/o mice
PrP staining near ischemic lesionsInfarct size, tetrazolium blue staining
David Westaway
Title goes here
“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
David Westaway
Title goes here
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
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