Prion Diseases

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					Prion diseases

Professor James W Ironside
National CJD Surveillance Unit & Division of Pathology
School of Molecular & Clinical Medicine
University of Edinburgh
Neurodegenerative disease and
aberrant protein deposition (1)
   Classical neuropathological investigation of
    neurodegenerative diseases have demonstrated the
    presence of abnormal histological structures, which
    have become the basis of diagnosis
   These include nuclear and cytoplasmic inclusion
    bodies and extracellular amyloid deposits
   Frequently these deposits are proteinaceous, fibrillar,
    and rich in b-pleated sheet secondary structure
   These observations have led to the idea that “fatal
    attractions” between abnormally folded forms of
    specific normal cellular proteins cause specific
    neurodegenerative diseases
Neurodegenerative disease and
aberrant protein deposition (2)
 Disease             Protein               Aggregates CNS region
 Alzheimer’s b-amyloid                     Extracellular           Hippocampus, cerebral
                                           plaques                 cortex
 Parkinson’s -synuclein Cytoplasmic   Substantia nigra
                         (Lewy bodies)
 ALS                 SOD1                  Cytoplasmic             Motor cortex, brain stem
 Huntington          Huntingtin            Variable                Striatum, cerebral
                                           intracellular           cortex
 SCA1                Ataxin-1              Nuclear                 Cerebellum, brain stem

 Prion               PrP                   Peri-, and              Various depending on
                                           extra-cellular          form of disease
Adapted from Kaytor and Warren (1999) J. Biol. Chem. 274:37507-37510
Prion diseases
   Rare, fatal neurodegenerative diseases
   Affecting humans (CJD) and agricultural, zoo and
    wild animals (scrapie, BSE).
   Genetic, sporadic and infectious aetiologies
   Can have lengthy incubation periods (> 40 years)
   Clinically characterised by dementia and ataxia
   Pathology characterised by neuronal loss, gliosis and
    spongiform change in the brain.
   No classical host response
   Also known as transmissible spongiform
    enecephalopathies (TSE)
Characteristic TSE Neuropathology

                           Aguzzi et al 2001
Animal TSE
Disease                Species                 Aetiology
Scrapie                Sheep and goats         Acquired (route
                       (Widespread, endemic,   unknown)
                       known since 18 cent)

Transmissible mink     Farmed mink (USA,       Acquired (route
encephalopathy (TME)   since 1940s)            unknown)

Chronic wasting        Elk and deer (USA,      Acquired (route
disease (CWD)          captive 1940s, wild     unknown)
                       population 1980)

Bovine spongiform      Cattle and exotic       Acquired (Probable
encephalopathy (BSE)   unglulates (Primarily   BSE contaminated
                       UK, reported 1987)      feed)

Feline spongiform      Domestic and large zoo Acquired (Probable
encephalopathy         cats (Primarily UK,    dietary exposure to
                       reported 1991)         BSE)
Human TSE (1)
Disease                   Incidence             Aetiology
Sporadic Creutzfeldt-     One per million per   Idiopathic
Jakob Disease             annum world-wide,

Familial Creutzfeldt-     Around 15% of all     Hereditary, autosomal
Jakob Disease             human TSE (total)     dominant

Gerstmann-Straussler-        ‘’                     ‘’
Scheinker Disease

Fatal Familial Insomnia      ‘’                    ‘’
Human TSE (2)
Disease             Incidence             Aetiology
Kuru                Fore tribe of New     Acquired (Ritual
                    Guinea (most common   canabalism)
                    cause of death in

Iatrogenic          France, Japan, UK,    Acquired:
Creutzfeldt-Jakob   USA (273)             Corneal transplant (4),
Disease (iCJD)                            Stereotactic EEG (2),
                                          Neurosurgery (5),
                                          Dura mater graft (120),
                                          Cadaveric human
                                          hormone therapy (147)

Variant             Since 1996            Acquired (Probable
Creutzfeldt-Jakob   UK (158)              dietary exposure to
Disease (vCJD)      Overseas 21           BSE)
                                          Blood transfusion (1)
Historical developments
   1936: Scrapie shown to be transmissible
   1954: Sigurdsson postulates “slow virus” causative agent
   1950s: Kuru associated with local practices rather than genetics
   1959: Hadlow recognizes similarities between scrapie and Kuru
   1966: Kuru transmitted to non-human primates
   1966: Alpers proposes a TSE agent devoid of nucleic acid
   1967: Griffith suggests that the TSE agent may be a protein
   1968: CJD transmitted to non-human primates
   1981: GSS transmitted to non-human primates
   1982: Protease-resistant protein (PrPSc) associated with infectivity
   1982: Prusiner suggests a novel proteinaceous particle (prion)
   1985: PrP found to be encoded by host genome (PRNP)
   1989: GSS linked to mutation in PRNP
   1991: Cellular PrPC is the conformational precursor of PrPSc
Prion hypothesis
   “Prions are unprecedented infectious pathogens that
    cause a group of fatal neurodegenerative diseases
    by an entirely novel mechanism”
   “Prions are transmissible particles that are devoid of
    nucleic acid and seem to be composed entirely of a
    modified protein (PrPSc).”
   “The normal, cellular PrP (PrPC) is converted into
    PrPSc through a post-translational process during
    which it acquires a high b-sheet content.”
   “In contrast to pathogens carrying a nucleic acid
    genome, prions appear to encipher strain-specific
    properties in the tertiary structure of PrPSc.”
                                                     Prusiner 1998
Aetiology of human TSE
according to the prion hypothesis
   Sporadic: Somatic mutation or chance conversion of
    PrPC to PrPSc
   Familial: Presence of mutations in the gene which
    encodes PrP (PRNP) that destabilize PrPC and
    predispose it to chance conversion to PrPSc
   Acquired: Exposure to exogenous PrPSc
    – Kuru: Oral exposure to human prions
    – Iatrogenic CJD: Medical exposure to human prions
    – vCJD: Oral exposure to bovine prions
Unresolved Questions
   What is the exact nature of the infectious agent?
   How does agent replication occur?
   How can we explain phenotypic diversity?
   What is the biochemical correlate of agent strain?
   What is the pathogenic (neurotoxic) mechanism?
PrP C     Structure
   PRNP encodes a 253 amino acid primary translation
   22 amino acid endoplasmic reticulum signal peptide
   23 amino acid C-terminal GPI anchor sequence
   N-terminus (23-121) largely unstructured, but
    contains the Cu2+-binding octarepeat region (51-91)
   C-terminus globular domain containing three -
    helices and two short b-pleated sheets
   N-linked glycosylation of variable occupancy at
    Asparagine 181 and 197
   The structure is stabilized by a disulphide bridge
    connecting helices 2 and 3
3D structure of   PrP C

                          Zahn et al 1999
Glycosylated         PrP C

occurring cellular
PrP is a
glycoprotein with
two bulky N-linked
glycans attached
to asparagines
181 and 197.
                                                         Prusiner 1998

Properties of PrPC and PrPSc

                    PrPC      PrPSc
  Normal cellular isoform     Disease-associated isoform
           3% beta sheet      43% beta sheet
               Monomeric      Highly aggregated
                   Soluble    Insoluble
Protease sensitive (PrPsen)   Partially protease resistant (PrPres)
PrPSc structure by electron

                              Wille et al 2002
  Post-translational conformational

Soto 2004
Cell free conversion

   Monomeric -helical PrP can be converted to a
    monomeric b-sheet form that is fibrilogenic and
    partially protease resistant by:
    –   Reduction of the disulphide bond and acidic pH
    –   Denaturation/renaturation with SDS
    –   Chaotropic salts at low pH
    –   Loading with divalent cations (Cu and Mn) and aging
        (associated with deamidation Asn107Asp)
Cell free conversion: template
   Mixture of 35S-labelled PrPC from cultured cells and
    PrPSc from infectious sources
   Measure formation of 35S-labelled PrPres
   Inefficient requiring excess PrPSc and converting only
    a small proportion of PrPC
   Efficiency improved by:
    –   denaturants
    –   crude cell lysates
    –   molecular chaperones
    –   sonication
Cellular co-factors & conversion:
Molecular chaperones
                   Importance of cellular
                   factors (eg chaperones
                   such as HSP104)
                   affecting PrPC substrate

                                   DebBurman et al 1997
Cellular co-factors & conversion:
Mammalian RNA
Mammalian brain
contain RNA that
stimulate the
conversion of PrPC
to PrPSc in a
modified PMCA

(Deleault et al, Nature
Synthesis and trafficking of PrP

                            Harris 1999
Life-cycle of           PrP C

 Rapid endocytic
 recycling of PrPC in
 cells (60 minutes)

                                Harris 1999
Conversion in cultured cells

                           Harris 1999
                                 Harris 1999

Aggregation and acquisition of


Model for conversion in situ

                          Harris 1999
Multiple conformations of                        PrP Sc?

   “In contrast to pathogens carrying a nucleic acid
    genome, prions appear to encipher strain-specific
    properties in the tertiary structure of PrPSc.”

   Is there evidence for heritable structural diversity in
    different prion diseases?
 PrPres Isotype by Western blot
Treatment with
proteinase K results
in N-terminal
truncation of PrPres

Distinct isotypes of
PrPres characterize
different forms of CJD

Isotypes differ in
extent of truncation
and degree of
glycosylation site
Codon 129 and   PrP res   isotype

                              Parchi et al 2000
          Glycotype complexity
Diversity of the
attached glycan may
also contribute to
disease phenotype
and agent strain

2D gel profiles from
two forms of MM2
sporadic CJD differ
dramatically in the
composition and/or
structure of the
attached glycans
                                 Pan et al 2001
Do different PrPSc types replicate
with fidelity in vitro?
When human PrPC
is converted to
PrPSc in a PMCA
reaction the
product has both
the conformation
and the
glycosylation ratio
of the in-put PrPSc

                           Soto et al, in press
Probing prion protein
conformation using CDI

   Fragment analysis by Western blotting of proteinase
    K treated prion protein is a very indirect and rather
    blunt instrument for examining conformation

   Conformation dependent immunoassay (Safar et al,
    Nature Medicine 1998;4:1157-1164) offers a more
    direct method for detecting differences in
Conformation Dependent
Immunoassay (CDI)

   Avoids proteinase K digestion
   Uses increasing concentrations of a chaotropic salt
    (GdnHCl) to unmask an epitope (108-111) buried in
    native PrPSc
   Produces a “melt curve” describing the unfolding
    kinetics of prion protein
   CDI was able to ascribe unique conformations to
    eight different scrapie strains
CDI analysis of hamster scrapie

                            Safar et al 1998
Conservation of          PrP

following transmission to mice
Inoculum         Host     Host PRNP         PrPres (kDa)

None             Human    FFI(D178, M129)          19

FFI              Mouse    Tg(MHu2M)                19

FFI Tg(MHu2M)    Mouse    Tg(MHu2M)                19

None             Human    fCJD(E200K)              21

fCJD             Mouse    Tg(MHu2M)                21

fCJD Tg(MHu2M)   Mouse    Tg(MHu2M)                21

                                              Telling et al 1996
Conservation of targeting
following transmission to mice
 FFI transmitted to
 Tg(MHu2M)Prnp0/0 mice
 Thalamic pathology

 fCJDE200K transmitted to
 Tg(MHu2M)Prnp0/0 mice
 Cortical pathology

 Telling et al 1996
Aspects of PrPSc structure that
might encipher strain properties
   Extent of structural re-arrangement (conversion to b-
    sheet) at the N-terminus.
   Presence of methionine or valine at codon 129
   Presence or absence of bound divalent cations
   Extent of of asparagine-linked glycosylation site
   Composition and complexity of attached glycans
Evidence for toxic gain of
function in PrPSc: The PrP106-126
in vitro model system
   General temporal and spatial correlation between
    PrPSc accumulation and pathological change
   PrPSc toxicity can be modeled in vitro using a
    synthetic fragment of PrP, termed PrP106-126
   Newly synthesized PrP106-126 is not toxic to cultured
   On aging PrP106-126 spontaneously adopts a b-sheet
    conformation, aggregates and acquires cytotoxic
    Effects of        PrP 106-126       on neurones
   Toxicity dependent on cellular PrPC expression
   Production of aggregated, mildly protease-resistant
    PrP isoforms
   Decreased antioxidant defenses
     – Bcl, GSSH, PrP-dependent SOD
   Altered membrane fluidity
     – Amyloid channels and unregulated calcium influx
   Pro-apoptotic changes
     – p38 MAP kinase dependent apoptosis
   Toxicity potentiated by the presence of PrP106-126
    treated microglia
Neurodegenerative mechanism

                          Hope 2000
Synthetic mammalian prions?

   Recombinant
    MoPrP (89-230)
    expressed in E.coli
    and purified
   Amyloid fibrils
    produced by urea
   Inoculated ic into
    Tg mice over-
    MoPrP(89-230) by
    a factor of 16
                          Legname et al, Science 2004;305:673-676
Synthetic mammalian prions?
   Tg mice developed
    neurological dysfunction
   Brains showed a
   Protease-resistant PrP
    was present
   Disease was
    transmissible by ic route
    to wild-type mice
   Novel prion strain
                                Legname et al, Science 2004;305:673-676
Proteins with prion-like
properties in other systems
   A prion-like switch in conformation appears
    to be the mechanism in the dominant
    cytoplasmic inheritance of certain genetic
    traits in yeast (Wickner, Science 1994;264:566-569)
   Epigenetic regulation of long-term
    potentiation by a prion-like switch in the
    neuronal cytoplasmic polyadenylation
    element binding protein, CPEB (Si et al, Cell
    2003;115:897-891, Si et al, Cell 2003;115:893-904)
Suggested further reading:
   Aguzzi A, Polymenidou M. Mammalian prion
    biology: one century of evolving concepts. Cell
   Soto C. Diagnosing prion diseases: Needs, challenges
    and hopes. Nat. Rev. Microbiol. 2004;2:809-819
   Ma et al. Neurotoxicity and neurodegeneration
    when PrP accumulates in the cytosol. Science
   Deleault NR, et al. RNA molecules stimulate prion
    protein conversion. Nature 2003;425:717-720
   Si K, et al. A neuronal isoform of the Aplysia CPEB has
    prion-like properties. Cell 2003;115:879-891
   Legname G, et al. Synthetic mammalian prions.
    Science 2004;305:673-676

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