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 Animals Scrapie (sheep & goat) Bovine spongiform encephalopathy (BSE, a.k.a. Mad Cow) Chronic wasting disease (CWD; deer & elk) Humans Kuru Creutzfeldt-Jakob disease (CJD)

Gajdusek and Alpers (1965) Kuru: A TRANSMISSIBLE encephalopathy Spongiform lesions

What is the causative agent of TSEs? Agent could pass through 0.2  m filter  NOT a bacterium (bacteria are usually  m) What about Protein? Agent could resist ionizing and UV radiation  NOT a nucleic acid-based agent (not a virus)  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 PrP C (C for cellular) is the normal cellular form of prions PrP Sc (Sc for scrapie) is the misfolded form PrP C can adopt the alternatively folded form, PrP Sc, which causes disease

Structural Characteristics of PrP C and PrP Sc The amino acid sequences of PrP C and PrP Sc are identical, but the proteins differ in their three-dimensional conformation PrP C – rich in  -helixes and little β-sheet PrP Sc – less rich in  -helixes and much more β-sheet structure PrP C PrP Sc

Prion conformational conversion PrP Sc acts as a template on which PrP C is refolded into the infectious disease-causing PrP Sc form PrP C PrP Sc

PMCA: protein misfolding cyclic amplification Mix small quantities of PrP Sc with excess of PrP C PrP Sc aggregates during incubation Sonication breaks PrP Sc aggregates into small pieces; this acts to multiply the number of “converting units” Repeat to get detectable levels of PrP Sc + PrP C PrP Sc sonicate incubate Serially repeat Adapted from Trends in Neuroscience, 2002 Aug;25(8):390-4.

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 PrP Sc PK-resistant C-terminal core insolubility tendency to aggregate ability to propagate a PK-resistant conformation to endogenous PrP C

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 Ban on meat and bone in cattle feed Epidemiological studies 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

BSE in cattle vCJD in humans Relationship of BSE and variant CJD 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.

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 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

Scenario On what information do you base your recommendation? 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. What additional information/data would you still like?

“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.” Centers for Disease Control and Prevention (CDC) Recommendation

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Extra slides

Surveillance in Wisconsin since deer tested 1576 positive (≈1%) Prevalence increases with age of deer CWD in Wisconsin deer (WI)

Australia 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)

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) Kuru: A TRANSMISSIBLE encephalopathy Spongiform lesions