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Transmissible Spongiform Encephalopathies
(TSEs)
a.k.a. Prion Diseases
Transmissible  can be spread
Spongiform  resembling a sponge
Encephalopathies  disorders/diseases of the brain
Characteristics of TSEs
• Cause insomnia, weight loss, ataxia (loss of
coordination), memory loss, dementia, death
• Incubation time ranges from 1-60 years
• Transmissible
• Invariably fatal
Spongiform lesions
in the brain
Examples of TSE Diseases
Humans
Animals
• Kuru
• Creutzfeldt-Jakob
disease (CJD)
• Scrapie (sheep & goat)
• Bovine spongiform
encephalopathy (BSE,
a.k.a. Mad Cow)
• Chronic wasting disease
(CWD; deer & elk)
Kuru: A TRANSMISSIBLE encephalopathy
Spongiform lesions
Gajdusek and Alpers (1965)
Spongiform lesions
What is the causative agent of TSEs?
• Agent could pass through 0.2 m filter
 NOT a bacterium (bacteria are usually 0.5-5 m)
• Agent could resist ionizing and UV radiation
 NOT a nucleic acid-based agent (not a virus)
• What about Protein?
Infectious agent purified by Stanley
Prusiner in 1982
identified as PrP, or prion protein (Nobel
Prize, 1997)
The Prion Hypothesis of TSEs
The infectious agent is a misfolded protein (PrP, or
prion) that propagates in the absence of nucleic acid
by transmission of its altered and pathogenic
conformation to the normal host version of the
protein.
Direct experimental evidence in support of this
hypothesis has been difficult to obtain
Prion terminology
• Prion proteins (PrP) are encoded by the host genome
• PrPC (C for cellular) is the normal cellular form of prions
• PrPSc (Sc for scrapie) is the misfolded form
• PrPC can adopt the alternatively folded form, PrPSc,
which causes disease
Structural Characteristics of PrPC and PrPSc
• The amino acid sequences of PrPC and PrPScare identical, but
the proteins differ in their three-dimensional conformation
• PrPC – rich in -helixes and little β-sheet
• PrPSc – less rich in -helixes and much more β-sheet structure
PrPC
PrPSc
Prion conformational conversion
PrPSc acts as a template on which PrPC is refolded
into the infectious disease-causing PrPSc form
PrPC
PrPSc
PMCA: protein misfolding
cyclic amplification
PrPC
+
•Mix small quantities of PrPSc with
excess of PrPC
•PrPSc aggregates during incubation
sonicate
•Sonication breaks PrPSc aggregates
into small pieces; this acts to multiply
the number of “converting units”
incubate
•Repeat to get detectable levels of
PrPSc
Serially repeat
Adapted from Trends in Neuroscience, 2002 Aug;25(8):390-4.
PrPSc
In-class assignment
Figure 1A.
Multiple rounds of PMCA using recombinant PrP in the
presence of synthetic anionic phospholipid (POPG) and RNA
results in a PK-resistant band appearing after 17 rounds.
Figure 1B.
The PMCA product generated in Figure 1A is highly resistant to
PK (a defining feature of misfolded, infectious prion proteins).
Figure 1C.
The PK-resistant species is found in the pellet (not supernatant)
after centrifugation, and results in a 15KDa PK-resistant band at
the C-terminus (insolubility, aggregation, and PK-resistant core
at C-terminus are defining features of misfolded, infectious
prion protein).
Figure 1D.
PMCA seeded with rPrP-res produced PK-resistant PrP bands in
normal mouse brain homogenate whereas unseeded PMCA did
not.
Figure 1E.
Infection of a mouse neuronal cell line with rPRP-res generated in
PMCA results in a PK-resistant PrP band (Numbers indicate cell
passages after infection).
What is the overall purpose of showing the
series of experiments in Figure 1?
To demonstrate that PMCA of bacterially-expressed PrP using
RNA and POPG generated a PrP protein with all the
characteristics of a PrPSc
• PK-resistant C-terminal core
• insolubility
• tendency to aggregate
• ability to propagate a PK-resistant conformation to
endogenous PrPC
Table 1.
Intracerebral inoculation of rPrP-res generated by PMCA with
POPG and RNA invariably caused disease in mice, demonstrating
that the misfolded protein produced from recombinant PrP is
infectious and pathogenic.
Do the results of this paper support or
contradict the “Prion Hypothesis”? Why?
While the efficient in vitro production of PrP-res required
the addition of cofactors, the fundamental principle of the
Prion Hypothesis is supported by this data, in that the
disease is transmitted by a misfolded protein.
Bovine spongiform encephalopathy
(BSE)
• “Mad Cow Disease” epidemic (1986-early 2000s)
• # of infected cattle estimated at > 1 million
• Spread by cattle being fed “meat and bone meal”
produced from infected cattle
The emergence and control of BSE
First case
Epidemiological
studies
Ban on meat
and bone in
cattle feed
BSE transmission
to mice in lab
Human Creutzfeldt-Jakob disease
(CJD)
• CJD is a human TSE
• Sporadic CJD
• 85% of CJD cases
• Average onset at 52 years of age
• Familial CJD
• 10-15% of CJD cases
• Average onset at 60 years of age
• Variant CJD, first reported in 1996
• <1% of CJD cases
• Average onset at 28 years of age
• Distinct pathological and clinical features
Relationship of BSE and variant CJD
BSE in cattle
vCJD in humans
•Association in
-Time
-Place (UK)
•>200 human cases
•Peak has past
•4 cases diagnosed in
2010
What have we learned from the BSE and vCJD
outbreaks?
• Prion diseases can cross species barriers to infect
humans
• MANY humans exposed, but few infected
• The majority of individuals who died of vCJD via
contaminated beef had a mutation in their native PrP
• Same mutations are found in
sporadic and familial CJD cases
• PrP mutation likely made cellular
PrP more susceptible to misfolding
Scenario
You are a public health official and receive a phone call
from a deer hunter in Wisconsin. He will be hunting for
deer this season, but has heard some grumblings among
friends about Chronic Wasting Disease (a transmissible
prion disease) in Wisconsin deer herds.
He is concerned about the potential risk associated with
preparing and eating deer meat and wants to know if he
should be concerned and what he can do to reduce the
risk.
Take one minute and write two things that we should
consider to assess the potential risk to this person.
Chronic Wasting Disease (CWD)
• A prion disease that affects deer, elk, and moose
• Highly transmissible within deer and elk populations
• Identified in 13 states and 2 Canadian provinces
Confirmed Cases of CWD in the U.S.
Chronic Wasting Disease (CWD)
•Spreads through the deer population by:
-animal-to-animal contact (prion shedding into saliva)
-contaminated feed and water sources
•No evidence of CWD transmission to humans
has been reported
•Surveillance has not shown any increase in the incidence of
Creutzfeldt-Jakob disease
•Efficiency of CWD to convert recombinant human PrP into
aberrant form is low, but similar to that of BSE and scrapie
Scenario
You are a public health official and receive a phone call
from a deer hunter in Wisconsin. He will be hunting for
deer this season, but has heard some grumblings among
friends about Chronic Wasting Disease (a transmissible
prion disease) in Wisconsin deer herds.
He is concerned about the potential risk associated with
preparing and eating deer meat and wants to know if he
should be concerned and what he can do to reduce the
risk.
On what information do you base your recommendation?
What additional information/data would you still like?
Centers for Disease Control and Prevention (CDC)
Recommendation
“Hunters should consult with their state wildlife agencies to
identify areas where CWD occurs and take appropriate
precautions when hunting in such areas. Hunters and others
should avoid eating meat from deer and elk that look sick or
that test positive for CWD. Hunters who harvest deer or elk
from known CWD-positive areas may wish to consider having
the animal tested for CWD before consuming the meat
(information about testing is available from most state
wildlife agencies). Persons involved in field-dressing
carcasses should wear gloves, bone-out the meat from the
animal, and minimize handling of the brain and spinal cord
tissues.”
End.
Extra slides
CWD in Wisconsin deer
• Surveillance in Wisconsin since 2002
• 166710 deer tested
1576 positive (≈1%)
• Prevalence increases
with age of deer
(WI)
Kuru: Epidemiology and Fore culture
• Identified in Fore linguistic group of
Papua New Guinea in 1954
• Fore practiced cannibalism as a form
of mourning
• Cessation of cannibalistic rituals led
to a decline in disease (last case in
2005)
Australia
Kuru: A TRANSMISSIBLE encephalopathy
• Gajdusek and Alpers collected brain tissue of
deceased 11-year-old Fore girl in mid 1960s
• Injected into two chimps (1965)
– One died 2 years later with identical symptoms
– First proven case of infectious encephalopathy
– Nobel Prize in Medicine (1976)
Spongiform lesions
Spongiform lesions