Prion Diseases

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Prion Diseases
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Kuru
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Kuru figures

• Incidence 1 (population aprox. 15,000)
• total F M 10 1
• lt 20 years of age F M 3 1
• (20 of cases)
• 20-40 years of age F M 15 1

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Etiology of Kuru

• Primary infectious no fever, no CSF cell raise
• Genetical disease
• Pro in families, in patients who moved out of
  Fore region.
• Con also in patients who came to live in Fore
  region, as well in young as in old patients.
• Toxic/deficiency no toxic compound isolated,
  balanced diet.
• Endocanibalism

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Sporadic CJD
1920/1921
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Sporadic Creutzfeldt-Jakob disease diagnosis

• Rare disease 1/106
• Duration short (months)
• Neurological signs and symptoms
• Rapidly progressive dementia, myoclonus,
  ataxia, central visual disturbances,
  extrapyramidal signs, pyramidal signs, akinetic
  mutism (variant chorea, dysesthesias,
  psychiatric disturbances)
• EEG and MRI
• Neuropathology
• WHO criteria
• Type of CJD (sporadic, genetic, iatrogenic,
  variant)
• Strength of diagnosis (definite, probable,
  possible)

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MRI
EEG abnormalities
normal
sCJD
Weighted T2
Proton density
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Gross sCJD
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Micro sCJD
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Prion Diseases/Transmissible Spongiform
Encephalopathies
Prion diseases
TSE's in animals
TSE's in man
sporadic
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sCJD
Scrapie
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genetic
fCJD
ex-Scr
acq. by infection
GSS
CWD
FFI
TME
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Kuru
BSE
iCJD
FSE
ex-TSE
vCJD
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Etiology

• Possibilities (ca 1980)
• Degenerative/Hereditary
• (slow)Virus

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Againts virus hypothesis

• Very resistant agent
• resists dry heat ( gt 200ºC)
• resists steam autoclaving (up to134ºC, 18 mins.)
• resists formaldehyde
• resists UV-radiation
• resists Gamma-radiation ( gt 0.3 MGray)
• etc.

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Prion docterine (Prusiner)

• In biochemical separation-infection studies
• Infectivity is not present in a DNA/RNA fraction
• Infectivity is present in a protein fraction
• In conclusion
• A protein (and that protein only) causes a prion
  disease

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From Prion gene to Prion protein
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PrPC
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PrPC to PrPSc
Abnormal folding of a protein protein (and that
protein only) causes a prion disease
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Conversion models for PrPC to PrPSc
and subsequent breaking and seeding.
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Structural assembly of PrPSc
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Aggregation
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Crystal /Scrapie associated fibril
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IHC for PrP in sCJD-neocortex
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The Z family
I
II
III
IV
Clin dementia, ataxia, hypokinesia
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Example of gCJD in cerebellum
Dominant inheritance with dementia and ataxia
mostly
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Familial CJD vs Familial Fatal Insomnia
178Asn-129V-f-CJD
178Asn-129M-FFI
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Prion diseases acquired through infection

• Kuru
• iatrogenic CJD
• new variant CJD

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iCJD
number
incubation time
clinical presentation
Intracerebral cornea neurosurgery
stereotactic EEG Cerebral surface dura
mater Peripheral blood growth hormone
gonadotrophin
4 5 2 174 180 4
17 mo 17 mo 18 mo 6-12 y 12 y 13 y
dementia/ataxia dementia/ataxia dementia/ataxia a
taxia ataxia ataxia
Mostly Lyodura, now 2 cases with 0.1 N NaOH
treated Tutoplast
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Bovine Spongiform Encephalopathy
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BSE medulla oblongata
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BSE-microscopy
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Clinical history in v-CJD

• 65 of cases onset with psychiatric complaints
• Behavioural disturbance
• Attention deficit
• Personality changes
• Depression
• Later weeks - months
• Dysaesthesia (20 onset with dysaesth)
• Ataxia
• Myoclonus/choreo-athetosis
• Progressive dementia
• No typical EEG
• 14-3-3 protein 50 of the cases positive
• bilateral Pulvinar sign on MRI

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MRI
Flair sCJD
Flair vCJD
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Age Comparison with sp-CJD
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vCJD autopsy
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Tonsil biopsies in vCJD
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Variant CJD
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vCJD caused by BSE?
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Strain typing

• Glycoform pattern
• Genotype/polymorphisms
• Clinical profile
• Inoculation in experimental animals
• Incubation time
• Pathology profile

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CH sidechains of PrP
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Glycoform analysis
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Survival after inoculation
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Lesion profiling example
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Lesion profiling
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Whats the problem with Prion diseases

• Infectious material (whatever the origin
  sporadic, genetic or infectious derived)
• Poorly disinfectable
• formic acid 96 1 hr
• 2 N NaOH 1 hr
• 20,000 ppm Chlorine 1 hr

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Smallest infectious dose
ID50/kD
kD
Smallest infectious dose 300-600 kD 14-28
PrPSc molec.
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Pathogenesis of infection

1. Direct brain contact
2. Vascular inoculation
3. Oral (intestinal) inoculation

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Role for autonomic nervous system

• 1997 infectivity after oral challenge in hamster
  first seen at T7 level of spinal cord (Diringer)
• 2002 PrPSc seen in vagus nerve and splanchnic
  nerve before reaching spinal cord after oral
  challenge (Beekes)

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Role for hematologic system

• 1996 immunodeficient mice develop no prion
  disease after peripheral inoculation
  (Bruce)-spleen dependency
• 1991 FDCs found positive for PrPSc in mice
  (Kitamoto)
• 1997 crucial role for B-cells in CJD
  pathogenesis (Aguzzi)
• 1998 PrP expression in B-cells not necessarry
  for CJD pathogenesis (Aguzzi) exit B-cells

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Proposed route of peripheral infection
GALT
gt
Spleen
B-cell


Macroph. FDC
Macroph. FDC
M-cell
lymphatics
Symp NS
Xth CN
Gut
Spinal cord
Brainstem
Brain
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PrPSc pathogenesis loss or toxic gain of
function?

• PrP properties
• Proposed function ligand
• Cu2 binding (metallo-protein)
• Hydrophobic section PrPcmt

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PrP protein-copper binding sites

• Manlgcwmlvlfvatwsdlglckkrpkpggwntggsrypgqgspggnryp
  pqggggwgqphgggwgqphgggwgqphgggwgqphgggwgqgggthsqwn
  kpskpktnmkhmagaaaagavvgglggymlgsamsrpiihfgsdyedryy
  renmhrypnqvyyrpmdeysnqnnfvhdcvnitikqhtvttttkgenfte
  tdvkmmervveqmcitqyeresqayyqrgssmvlfssppvillisflifl
  ivg
•  
• Cu binding motiv hgggw or ggth

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Octarepeats
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Normal
Pathology
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Generation of H2O2
H0

• Cu 2 2PrP PrP2-Cu
• Shiraishi et al Biochem J 2005387 247-255

H20
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Built in of PrPSc in lipid rafts

• Transformation of PrPC to PrPSc occurs
  preferentially in cholesterol rich lipid rafts
  (Hooper Biochem Soc Trans 2005 32 335-338)
• Cell membranes have a role in transformation of
  PrPC to PrPSc (Kazlauskaite Pinheiro Biochem
  Soc Symp 2005 72 211-222)
• Prion replication alters the distribution of
  synaptophysin in membrane (Russelakis-Carneiro
  et al Am J Pathol 2004 165 1839-1848)

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Protein misfolding to neuronal dysfunction
pathogenesis

• Protein misfolding accumulation diseases
• Alzheimers disease
• Parkinsons disease
• Tauopathies
• Progressive Supranuclear palsy
• Fronto-temporal dementias
• Cortico-basal degeneration

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Side step Alzheimers disease

• Non infective disease
• Leading to dementia

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Amyloid Precursor Protein
Membrane
Extra cellular
Signal peptide
Glycosylation
Aß
671-713
Cystein rich area
Phosphorylation
daefrhdsgyevhhqklvffaedvgsnkgaiiglmvggvvia
ß - secretase
a - secretase
? - secretase
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ABeta generation
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Toxic oligomers in ABeta

• Cu 2 leads to H2O2 production
• A Beta density in AD is related to synapse loss
  (lower level of synaptofysin)
• Small oligomers more toxic than large aggregates
• Sounds familiar???

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Protein folding diseases
Protein-normal folded

Mutation in protein
Chaperones/Ubiquitin/ Clipping mechanisms
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Hydrophobic interactions
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age
Protein-abnormal folded small aggregates (toxic)
lipid raft synapse dysf
H2O2
Protein aggregated (non toxic?)
amyloid
membrane damage
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Break down of referred patientsApril 1,
1998-December 1, 2006
1322 referred
595 pending file
727 true referrals
32 info refused
17 pending classification
43 alive
635 referrals at study end-point
13 possible prion disease
332 Prion cases (279 definite 53
probable) Mean mortality 1.09 /106.yr 289 non
Prion (164 definite 125 probable)
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Incidence of CJD Deaths in Canada (per Million
Population by Year)
Average incidence rate of CJD in Canada 1.09
per million Canadians
2
3
13
18
24
31
35
30
36
29
42
40
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Classification of Referrals
Definite CJD 279 Definite sporadic CJD (sCJD) Definite sporadic CJD (sCJD) 256
Definite familial CJD (fCJD) Definite familial CJD (fCJD) 5
Definite GSS Definite GSS 11
Definite FFI Definite FFI 1
Definite sporadic GSS Definite sporadic GSS 1
Definite iatrogenic CJD (iCJD) Definite iatrogenic CJD (iCJD) 4
Definite variant CJD (vCJD) Definite variant CJD (vCJD) 1
Probable CJD 53 Probable sporadic CJD (sCJD) Probable sporadic CJD (sCJD) 49
Probable familial CJD (fCJD) Probable familial CJD (fCJD) 4
Possible CJD 13 Possible sporadic CJD (sCJD) Possible sporadic CJD (sCJD) 13
Non-CJD 289
TOTAL 635
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CJD Cases (n332)
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sCJD Cases by Province/Territory
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NORTHWEST TERRITORIES
BRITISH COLUMBIA 40 99
NEWFOUNDLAND 4 83
ALBERTA 27 84
SASKATCH- EWAN 15 161
MANITOBA 16 144
QUEBEC 68 97
ONTARIO 115 96
PRINCE EDWARD ISLAND 0
NOVA SCOTIA 11 124
NEW BRUNSWICK 10 141
Actual sCJD of expected CJD (based on 2006
population)
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Canadian iCJD cases (dura mater)
Incubation Period
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vCJD criteria in Canadian case
I A Progressive neuropsychiatric disorder
B Duration of illness gt 6 months
C No alternative diagnosis
D No iatrogenic exposure
II A Early psychiatric symptoms
B Painful dysaesthesias
C Ataxia
D Myoclonus or chorea or dystonia
E Dementia
III A No EEG/No classical EEG Generalized triphasic periodic complexes at approx. 1 per second
B MRI pulvinar sign positive
IV Positive tonsil biopsy for PrP
Possible vCJD only (without pathological
confirmation)
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Occipital Neocortex HE of Canadian Case
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Travel history

• Sep 87-Aug 90 UK education 2 weeks
  visit France.
• Feb 91-Mar 91 visit UK
• Feb 92-Mar 92 visit UK
• June 2000 visit UK
• Total 38 months risk exposure 1-14 years
• prior to symptoms

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Survival all types of CJD and non-CJD
duration of disease (mo) duration of disease (mo)
mean median
sCJD 6.3 0.3 4
fCJD 18.0 6.0 10
GSS 69.3 18.6 35
non-CJD 22.8 2.3 16
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Cases of which unsuspected cases
sCJD Definite 256 22 (8.6 )
Probable 49
GSS Definite 11 8 ( 73 )
Probable 0
fCJD Definite 5 0
Probable 4
iCJD Definite 4 0
Probable 0
vCJD Definite 1 0
Probable 0
Total 332 30 (9.0 )
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Why where they missed?

• Lack of experience?
• Different age of onset/duration?
• Different clinical signs and symptoms?
• Different subtype?
• Different auxiliary investigations findings?

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Conclusions unsuspected Prion disease cases-1

• 9 of Prion disease cases were clinically
  unsuspected in Canada in last 8½ years.
• GSS poorly recognized (73 of GSS are clinically
  unsuspected in this series)

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Conclusions unsuspected Prion disease cases-2

• sCJD and GSS can be missed clinically
• Missing is due to the fact that these cases are
  atypical
• Less usual subtypes (MM2, MV2)
• Missing symptoms in the presentation
• Longer duration of disease
• Or (occasionally) due to missed clues
• MRI T2/FLAIR attenuation
• EEG
• Missing is not due to academic status of
  clinician

...but...are we missing much more cases??
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Back of the envelope

• Minimum True Mortality rate of CJD for Canada
  1.09/106.yr
• Maximum True Mortality rate of CJD for Canada ?
  
• General autopsy rate in Canada 9.5 110.5
  1(9.5 1) means 9.5x(30/332)x0.91x1.09/106.yr
  unnoticed CJD as they are not autopsied actual
  CJD frequency in Canada could be max 1.09
  /106.yr 0.85/106.yr 1.94/106.yr
• Comparison
• European surveillance figures for Switzerland,
  Spain, Italy, France and Austria have been last 2
  or more years well over 1/ 106.yr, and even over
  2 in selected case.
• True mortality figure is probably somewhere
  between
• 1.09 and 1.94.

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Prion diseases- regional distribution in the
Netherlands until 2000 -
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Is sporadic CJD really sporadic?

• Concept of spontaneous generation of abnormal
  prion protein is difficult to grasp
• But no relation with food, surgery or other
  possible cause found
• However in UK geographical clustering seen 15-25
  years before onset