Download The New TSE


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

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!

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].”


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.

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.


“TSE agents breed true in various tissues, cell
cultures, and cross-species transmissions
whereas PrP patterns do not.”

What does this mean?

Main hypothesis: That PrP-res itself is not
infectious.

Attempting to establish the difference
between CJD Agent characteristics and PrP
characteristics.

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.
Original
Sample
Cell Culture
Reinoculation
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.”

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.

“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?]

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.

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.

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.

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.
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.
“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.


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.

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.

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.
 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?

(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

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?