The New TSERachel McCabe

Critical Distinction Laura Manuelidis argues a difference

between a TSE AGENT and a PrP PATTERN.

Her research uses new techniques to attempt to define the differences› PCR› Strict Controls› New Digestive Enzymes

Evidence presented in the Intro

Qualms with the original report claiming PrP is the infectious agent

Now, even protein-only supporters propose that there is an additional infectious entity › (Colby and Prusiner, 2011)

More tests need to be done with new technologies to measure the infectious titers!

Qualms with the original reports claiming PrP is the infectious agent

Analysis in the past (1983 PK digestion) showed very varied results› A <3 log protein loss yielded a 6 log loss of infectivity.› A 4 log reduction of protein yielded only an ~2 log loss

of infectivity.

“Remarkably, we could find only one other titration of an apparently complete PrP digestion. This showed only a very small 0.5 log reduction in infectivity that accompanied the disappearance of all PrP forms [Suzuki et al., 2008].”

Protein-only supporters propose that there is an additional infectious entity

From Prusiner’s 2011 Paper: PrPC may need to enter a partially unfolded, intermediate state to

interact with PrPSc and undergo conversion; this intermediate state is referred to as PrP*. The conversion of PrPC to PrPSc may also require the assistance of one or more as-yet-unidentified cofactors, provisionally designated protein X. Presumably, protein X binds to PrPC and enables it to interact with PrPSc for conversion. Overexpression of protein X would thus shorten incubation times for disease, whereas ablation of protein X would prolong or abolish prion disease. Many putative protein X genes have been identified, but transgenic knockouts for these genes have failed to alter incubation times substantially. Several in vitro investigations have suggested that polyanions, including nucleic acids, may accelerate prion formation although this has not been shown in animals.

More tests need to be done with new technologies

PK works as a digestion enzyme, but apparently, keratinases are more effective at highly ordered proteins and Beta-sheet structures. › They have been shown to reduce PrP-res to

undetectable levels. Removal of PrP allows us to see if there is any

other infectious agent. Experiment is adapted to test for many different

mouse-adapted TSE agents, including human derived vCJD, kuru, and FU-CJD, as well as sheep derived scrapie agents.

Point (II) “TSE agents breed true in various

tissues, cell cultures, and cross-species transmissions whereas PrP patterns do not.”

What does this mean?

Two Creutzfeldt–Jakob disease agents reproduceprion protein-independent identities in cell cultures (Arjona et al., 2004)

Main hypothesis: That PrP-res itself is not infectious.

Attempting to establish the difference between CJD Agent characteristics and PrP characteristics.

The Experiment Researchers used two strains of CJD:

› FU In vivo, has a fast incubation time, many more amyloid deposits

› SY In vivo, has a slower incubation time, fewer deposits.

The Experiment

The Results When the two strains were inoculated

in cell culture, there were not many distinct differences in PRP-res patterns:› Both the FU and the SY strains looked

identical when grown on the same cell line.

“the PrP-res bands from FU and SY in GT1–7 cells were the same in three independent analyses, again indicating the PrP-res pattern is cell-type- rather than agent-specific.”

Re-inoculation Though the PrP-patterns were identical,

when FU and SY were reintroduced to host animals, they maintained their initial pathologies!

Basically, the protein patterns looked the same, but the diseases still manifest differently, even after extensive subculture.

Discussion “No purified, recombinant, transgenic,

or amplified form of PrP-res has been capable of reproducibly transmitting infection.”

These are strong words… What evidence from the rest of this class would you use to counter it? › WWPD? [what would Prusiner do?]

Reciprocal Interference BetweenSpecific CJD and Scrapie Agents inNeural Cell Cultures (Nishida et al., 2005)

Remember talking about interference previously?

Hypothesis: Infection of mice with SY-CJD interferes with superinfection by the more virulent FU-CJD and does not require PrP-res.

Controls Neural cells, which can be susceptible

to TSE agents, would be incapable of producing many of the cytokines that can participate in strain interference in vivo.

Ensures that the only differing factor is the presence of SY.

Results Two scrapie strains eliciting the same PrP-

res band pattern show markedly different susceptibilities to the additional CJD agent.

Presence of SY prevented superinfection by other agents.

Protection was independent of the amount of PrP-res already present in the SY culture.

Results Interference did not depend on the

presence or absence of abnormal PrP, but it was correlated with the previous infection.

This supports the viral agent hypothesis because the development of CJD symptoms was not correlated with the presence/absence of PrP-res.

Explanation for CJD’s rarity? It is suggested that the protective

power of the slow form of vCJD could explain why so few people have developed the disease.

Could also explain the clustering of the disease around small areas: › Consistent with an environmental agent of

low virulence.

So, is it a virus?? “Infectious TSE particles have a discrete

viral size of 25 nm and 107 and releasing their tightly bound nucleic acids destroys infectivity. Thus, some TSE agents such as SY may produce defective interfering particles, as found in many persistent viral as well as noncoding human viroid infections.”

They measured this in 1992.

Strain-Specific Viral Properties of VariantCreutzfeldt–Jakob Disease (vCJD) Are Encoded bythe Agent and Not by Host Prion Protein (Manuelidis et al., 2009)

This is much of the same support as previously shown:

› Similar to viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species.

› The PrP expression does not correlate with infectivity.

The Experiment Passages of the human vCJD agent

showed that strain-specific characteristics were encoded by the agent, not by PrP.

› Again, the disease characteristics differed, but PrP-res patterns were similar.

Results One of the tenants of the prion hypothesis

was the species barrier: › Current prion theory states that PrP

converts itself into the infectious agent, so in order to have a short incubation time, it would require identical PrP sequences in the donor and recipient host

› This was used as a possible explanation for the low virulence.

Discussion Wild type mice injected with human

vCJD showed shorter incubation times than mice expressing only human PrP. › This finding is not in accord with the

idea of a protein only species barrier

› Didn’t we see contradicting data earlier?

Revisiting the initial point (ii) TSE agents breed true in various tissues,

cell cultures, and cross-species transmissions whereas PrP patterns do not. › Prion diseases maintain their pathology after

being transmitted through various hosts. › Somehow, there is a source of information that is

allowing this transmission. TSE strains maintain their identity

despite various changes in prion protein. This fact strongly implicates a relatively stable but mutable viral genome

Your Thoughts! Dr. Manuelidis has presented a lot of

data, and is very dedicated to the cause. › After this information, what do you think? › Do you think her claims are too broad?

Correlation vs. causation. › Why/why not should this data being taken

seriously by the prion research community?