Teresa J'T' Pinheiro

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Folding, aggregation and neurotoxicity of prions

• Teresa J.T. Pinheiro
• Department of Biological Sciences

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Neurodegenerative diseases of protein aggregation
Protein aggregation
Neuronal cell damage

• TSEs
• Scrapie
• BSE
• CJD kuru FFI GSS
• CWD
• Alzheimers disease
• Parkinsons disease
• Huntingtons disease

Degeneration of brain function
Death of patient
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CJD statisticsSurveillance Unit Edinburgh 2nd
October 2007

As at 28th September 2007
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BSE and vCJD statistics
UK population MV 51 MM 38 VV
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DEFRA BSE surveillance 28th September 2007
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Prion conversion and pathogenesis
PrPC
PrPSc
PrP fibrils
Clinical symptoms
Amyloid plaques
Spongiform degeneration
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The prion or protein-only hypothesis
Griffith JS (1967) Self replication and scrapie.
Nature 215 Prusiner SB (1982) Novel proteinaceous
infectious particles cause scrapie. Science 216
Stanley B Prusiner The Nobel Prize in Physiology
or Medicine 1997 for his discovery of Prions - a
new biological principle of infection
PrPSc
PrPC
Legname et al. (2004) Synthetic mammalian prions.
Science 305
7
The virion hypothesis
Manuelidis et al. (1987) Evidence that PrP is not
the infectious agent in CJD. EMBO J. 6 Manuelidis
et al. (1995) Virus particles are required for
infection in CJD. PNAS 92 Manuelidis (2006) A 25
nm virion is the likely cause of TSEs. J. Cell.
Biochem.
A 20-30 nm round or dodecahedral particle with a
protected viral genome of 1-4 kb capable of
encoding a nucleocapsid protein and/or an enzyme
necessary for viral replication.
Arjona et al. (2004) Two CJD agents reproduce
PrP-independent identities in cell culture. PNAS
101 Nishida et al. (2005) Reciprocal interference
between CJD and scrapie agents in cell cultures.
Science 310
Baruch S. Blumberg and D. Carleton Gajdusek The
Nobel Prize in Physiology or Medicine 1976 for
their discoveries concerning new mechanisms for
the origin and dissemination of infectious
diseases Unconventional Viruses and the Origin
and Disappearance of Kuru
Safar et al. (2005) Search for a prion-specific
nucleic acid. J. Virol. 79
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Can all self-replicating protein aggregates be
infectious?
Alzheimers disease Parkinsons disease Type II
diabetes Prion diseases and others
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Folding and prion pathogenesis
Interaction with lipid membranes conversion
nucleation and polymerisation of prions
In vitro folding of prion folding pathway
intermediates
Sporadic CJD 85 familial cases 10
transmitted lt 5
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In vitro (re)folding of the prion protein
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The folded C-terminal domain of PrP
I
II
N
N
S1
III
S2
C
C
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In vitro refolding of the truncated C-terminal
domain
David Jenkins
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Single-Trp mutants of PrP
F198W F175W
pH 5.5
U
N
pH 7.0
F198W F175W
U
N
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Structure of Trp mutants and reversible folding
PrPF175W
PrPF198W
PrPwt
PrPwt
U
U
F
F
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The elusive intermediate
PrPF175W
PrPF198W
pH 5.5 DGU(H20) 11 kJmol-1 D1/2 (CD) 4.4
M D1/2 (Trp) 3 M
a-helix
Trp
a-helix
pH 7.0 DGU(H20) 12 kJmol-1 D1/2 (CD) 5 M D1/2
(Trp) 3.5 M
Trp
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An intermediate in the folding of PrP
PrPW175 pH 5.5
PrPW175 pH 7.0
PrPW198 pH 7.0
PrPW198 pH 5.5
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The intermediate state has a high helix content
and forms fibrils
pH 5.5
N
U
I
200 nm
Jenkins et al. (2007) FEBS J. (in press)
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Conclusions

• I has a native-like secondary structure content
  ? diagnosis difficult
• Active conformation that can switch to the
  b-sheet aggregated state
• Details of the conversion unlikely to be via a
  monomeric b-sheet state but concomitant with
  oligomerisation

U ? I ? N
? PrPn ? PrPSc
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The protein folding universe

• Ageing of the cellular machinery point mutations
• Interaction with other cellular factors lipid
  membranes
• Impaired protein degradation
• Oxidative damage

Jahn Radford (2005) FEBS J.
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Aggregation of prions on membranes
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The Cellular Prion Protein(PrPC)
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Rafts in cell membranes
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Non-uniform distribution of PrPC on the cell
membrane
GT1-1 cells anti-PrP antibody SAF83
Sanghera et al. (unpublished)
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PrPC co-localises with GM1-rich domains on the
cell membrane
PrP
GM1
Merge
Permeabilised B104 cells anti-PrP antibody
SAF83 binding of conjugated cholera toxin
subunit B chain to GM1 ganglioside
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Prion trafficking and conversion
plasma membrane
ER
PrPC
trans Golgi network
secretory vesicle
nucleus
PrP
Golgi cisternae
PrPnuc
PrPSc
early endosome
lysosome
transport vesicle
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Brain isolates of PrPSc contain lipid
Kazlauskaite, Gill Pinheiro (unpublished)
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Structure of a- and b-Pr isoforms
CD
FTIR
b-PrP
b-PrP
a-PrP
a-PrP
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Binding of PrP to membranes

• Rafts
• DPPC, cholesterol, SM, gangliosides (GM1)
• Non-rafts
• charged lipids (POPG POPS)
• zwitterionic lipids (POPC)
• Membrane environment
• pH 7 (plasma membrane)
• pH 5 (endosomes)

Unilamellar lipid vesicle
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Calcein release
Membrane leakage
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Aggregation and fibrillization of PrP on lipid
vesicles
200 nm
200 nm
200 nm
Kazlauskaite et al. (2003) Biochemistry 42, 3295
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Structure, membrane effects and aggregation of
PrP in lipid membranes
Sanghera Pinheiro (2003) JMB Kazlauskaite et
al. (2004) Biochemistry Kazlauskaite et al.
(2005) BBRC
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Prion conversion on the membrane surface
PrPSc
PrP
PrPC
PrPnuc
raft
raft
plasma membrane
PrP
endocytic vesicle
Pinheiro (2006) Chem. Phys. Lipids
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Assembly of prion on membranes Phil Robinson
Membrane binding
Growth
Conformational change
Nucleation


Supported lipid membrane
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Nucleation and growth of prion plaques on
membranes
B
C
D
A
D
B
A
C
upper row 8 x 8 mm height 30 nm lower row 2.5
x 2.5 mm height 30 nm
Robinson, Reviakine Pinheiro (unpublished)
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Nucleation and growth of sponge-like prion
aggregates on membranes
C
A
B
25 x 25 mm height 70 nm
Robinson, Reviakine Pinheiro (unpublished)
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Prion promotes the formation of gap-like junctions
PrP
Cryo electron microscopy of vesicles of POPCPOPS
(82) with and without PrP
Robinson, Stoilova-McPhie Pinheiro
(unpublished)
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Conclusions and biological implications

• Lipid membranes may pay a role in prion
  conversion in vivo
• Prion conversion destabilises lipid membranes ?
  cell death possible mechanism of neurotoxicity

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The neurotoxic molecule in prion diseases
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Intermediates are more toxic than mature fibrils
Caughey Lansbury (2003) Ann. Rev Neurosci.
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In vitro prion conversion
b-sheet
a-helix
Kazlauskaite et al. (2005)
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Immunofluorescence labelling of PrPC on
permeabilised cells
A
B
C
GT1-1 Hypothalamic neurons Neurofilament 200
() Resting potential Action potentials Ca2
current
B104 Neuronal Neurofilament 200 (-) Na channels
N2a Unknown/neuronal Neurofilament 200
() Resting potential Action potentials
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The morphology of prion aggregates varies with pH
and time
Fresh
Aged
B
A
Globular aggregates
200 nm
200 nm
C
D
Pre-fibrils
200 nm
200 nm
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Conformation of PrP aggregates

• Globular aggregates have exposed side chains
• Pre-fibrils are more ordered and compact

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Cytotoxicity of b-sheet-rich aggregates of PrP
Fresh PrP
Aged PrP
a-syn
Glob PrP
Pre-fib PrP
Sanghera et al. (2007) BBA in press
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Where does exogenous PrP end up in exposed cells?
PrP
GM1
Merge
Labelling of exogenous rPrP with SHa-specific
anti-PrP antibody 3F4 CT-B binding to GM1
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Fate of exogenous PrP
PrP
NF
Merge
Exogenous PrP co-localises with intracellular
cytoskeleton protein neurofilament (NF) 200
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Effect of b-PrP on the electrophysiology of
neuronal cells
Control cells
Treated cells
Mark Wall
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Conclusions

• Globular aggregates and pre-fibrils of PrP are
  toxic
• Early neurotoxic effect of prion aggregates may
  involve alterations in the electrical properties
  of neurons

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Future work
Sanghera, Wall Pinheiro (2007) Curr. Contents
(in press)
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Warwick Imanpreet Bath Bruno Correia Joana
Correia Matthew Hicks David Jenkins Jurate
Kazlauskaite Phil Robinson Narinder Sanghera Ian
Sylvester Anna Young Richard Cowan Mridula
Swayampakula
2351 m
Collaborators Andrew Gill (IAH, Compton) Ted King
(TgK Scientific) Louise Kirby (IAH,
Compton) Julie Macpherson (Chemistry) Gerry Ronan
(Farfield Scientific) Catherine Vénien-Bryan
(Oxford) Svetla Stoilova-Mcphie (Warwick) Mark
Wall (Neurosciences) Mike Geeves
(Canterbury) Ilya Reviakine (San Sebastian)
Funding The Royal Society The Wellcome
Trust Medical Research Council Engineering and
Physics Sciences Research Council Biotechnology
Biological Sciences Research Council