SEARCH VEGSOURCE:

 

 

Follow Ups | Post Followup | Back to Discussion Board | VegSource
See spam or
inappropriate posts?
Please let us know.
  




From: TSS (216-119-143-93.ipset23.wt.net)
Subject: Autocatalytic self-propagation of misfolded prion protein [FULL TEXT]
Date: August 18, 2004 at 1:37 pm PST


Greetings,

i might remind some of you that some of these charactors/digits? are
not correct when i copy and paste this data from pdf to plain text.
i tried to correct a few but realized that my machine cannot do this.
(well, maybe my machine can, but i can't :-)
like 2 to the 9th power-fold came out 29-fold etc.
i thought it would still be helpful to some...TSS
=================================


Autocatalytic self-propagation of misfolded
prion protein
Jan Bieschke* , Petra Weber*, Nikolaus Sarafoff*, Michael Beekes!, Armin
Giese*, and Hans Kretzschmar*
*Center for Neuropathology and Prion Research, Ludwig Maximilians
University of Munich, Feodor-Lynen-Strasse 23, 81377 Munich, Germany;
and !Robert
Koch Institute, Nordufer 20, 13353 Berlin, Germany
Communicated by Manfred Eigen, Max Planck Institute for Biophysical
Chemistry, Go¨ ttingen, Germany, June 30, 2004 (received for review
March 1, 2004)
Prions are thought to replicate in an autocatalytic process that
converts cellular prion protein (PrPC) to the disease-associated
misfolded PrP isoform (PrPSc). Our study scrutinizes this hypothesis
by in vitro protein misfolding cyclic amplification (PMCA). In serial
transmission PMCA experiments, PrPSc was inoculated into healthy
hamster brain homogenate containing PrPC. Misfolded PrP was
amplified by rounds of sonication and incubation and reinoculated
into fresh brain homogenate every 10 PMCA rounds. The amplifi-
cation depended on PrPC substrate and could be inhibited by
recombinant hamster PrP. In serial dilution experiments, newly
formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed
the structural conversion of PrPC as efficiently as PrPSc from brain
of scrapie (263K)-infected hamsters, yielding an ~300-fold total
amplification of PrPres after 100 rounds, which confirms an autocatalytic
PrP-misfolding cascade as postulated by the prion hypothesis.
PrPres formation was not paralleled by replication of biological
infectivity, which appears to require factors additional to
PrP-misfolding autocatalysis.
Transmissible spongiform encephalopathies such as
CreutzfeldtJakob disease, bovine spongiform encephalopathy,
and scrapie are caused by unique transmissible agents
consisting of proteinaceous infectious particles, termed prions
(1). Prions are considered to consist mainly, if not solely, of a
misfolded scrapie-associated, aggregated, and proteinase Kresistant
isoform (PrPSc) of the cellular prion protein (PrPC) (2).
The infectious process is believed to follow an autocatalytic
mechanism in which PrPC is converted into the misfolded
infectious state by PrPSc. PrPC recruited and misfolded by
PrPSc subsequently catalyzes the conversion of further PrPC,
eventually leading to massive replication of infectivity and fatal
disease. To distinguish between PrPSc, which is isolated from
infectious tissue and is by definition associated with the transmissible
spongiform encephalopathy agent, on the one hand and
structurally altered PrP, which has been converted into a misfolded
proteinase K-resistant (PrPres) form in vitro but which
does not necessarily correlate with infectivity, on the other, we
defined the latter as PrPres.
For a long time, PrPC has been shown to acquire proteinase K
resistance in vitro under the impact of PrPSc (3). Saborio et al. (4)
reported a new experimental approach, protein misfolding cyclic
amplification (PMCA), for the generation of PrPres in vitro by
using brain homogenates taken from healthy Syrian hamsters
(SHas) mixed with PrPSc. In PMCA, the amount of PrPres
increases in a cyclic process of alternating incubation and
sonication steps. The amplification is assumed to follow a
mechanism of seeded aggregation in which the ultrasonic treatment
breaks the PrP aggregates into smaller units. These fragments
in turn then provide additional seeds for further aggregate
growth (5, 6). Amplification has also been observed without
sonication (7) or with a single sonication step (8) and seems to
depend on the presence of small RNA fragments (9).
In particular, the protein-only model of the prion hypothesis
implies that not only the original PrPSc but also newly formed
PrPres aggregates exert a converting activity on PrPC. According
to the data available so far (4, 7, 9), the generation of misfolded
PrP by PMCA would be consistent with such autocatalysis but,
alternatively, could also be explained by a simple catalytic
conversion of PrPC by the initial PrPSc present in the sample.
Thus, so far, whether PrPres generated in a PMCA reaction is
infectious (i.e., able to transmit disease in vivo), or, indeed,
possesses any catalytic activity to structurally convert PrPC at all
has not been demonstrated.
Therefore, the present study intends to further investigate
whether the PMCA conversion of PrPC to PrPres is mediated by
an autocatalytic mechanism, and models the replication of
infectivity according to the prion hypothesis. For this purpose,
we scrutinized the replication mechanism implied by the prion
hypothesis by addressing the following questions on PMCA. (i)
Does PrPres generation depend on the presence of a specific PrP
substrate? (ii) Can PrPres generation be specifically inhibited?
(iii) Does PrPres generated by PMCA serve as a template for
conversion of further PrPC to PrPres? (iv) Is PrPres generated by
PMCA infectious in animal experiments?
Methods
PMCA of PrPres. Normal SHa brain homogenate (10% wt/vol) was
prepared as described by Saborio et al. (4) and was spiked with
brain homogenate prepared from terminally ill hamsters intracerebrally
(i.c.) infected with 263K scrapie. Samples of 200 ul
were subjected to 23 amplification rounds consisting of 5 x 1 s
sonication with a ultrasonic microtip probe at 40% power setting
(Sonopuls 2070, Bandelin, Germany) followed by 1 h of incubation
at 37°C. The probe tip was washed between samples with
NaOH (2 M) and water to remove prions adherent to the metal
surface (10). Control samples were frozen immediately or were
incubated for the duration of the experiment at 37°C. After 10
rounds and 23 PMCA rounds, aliquots of 20 u1 were frozen and
stored at -80°C.
PrP0/0 brain homogenate was prepared as above from Prnp0/0
mice (11). For inhibition experiments, hamster recombinant PrP
(rPrP) (23231) (Prionics, Schlieren, Switzerland), mouse rPrP
(23231) prepared from Escherichia coli according to ref. 12, or
BSA (Sigma) was added before the PMCA reaction at final
concentrations of 7.5250 nM. Concentrations of rPrP were
determined by UV absorption measurement, and relative levels
of rPrP and PrPC were determined by comparing Western blot
band intensities of a rPrP dilution series with the following band
intensity of PrPC from hamster brain homogenate after deglycosylation
with PNGase F (New England Biolabs) according to
the manufacturers instructions (data not shown).
In PMCA with serially diluted PrPres, the amplification
procedure was carried out as above. After each amplification
cycle consisting of 10 rounds of alternating sonication and
Abbreviations: PMCA, protein misfolding cyclic amplification; PrP, prion
protein; PrpC,
cellular PrP; PrPSc, scrapie-associated PrP isoform; PrPres, proteinase
K-resistant PrP; LD50i.c.,
50% intracerebral lethal dose; rPrP, recombinant PrP; SHa, Syrian hamster.
To whom correspondence should be sent at the present address: The
Scripps Research
Institute, BCC 265, 10550 North Torrey Pines Road, La Jolla, CA 92037.
E-mail: jbiesch@
scripps.edu.
© 2004 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0404650101 PNAS  August 17, 2004 
vol. 101  no. 33  1220712211
BIOPHYSICS

incubation, the reaction mixture was diluted 1.5- 2-, or 2.5-fold
into normal hamster brain homogenate. In total, 10 amplification
cycles (IX in Fig. 3) with nine dilution steps were performed.
Samples of 20 u1 were taken before and after each round
and archived at -80°C. At the end of the experiment, samples
were thawed for Western blot analysis and bioassay (compare
Fig. 4). The sample obtained after ten passages with 2-fold
dilution was used in bioassay titration (sample B). Control
samples were frozen immediately (sample A) or after 29-fold
(sample C) dilution in 10% (wt/vol) normal hamster brain
homogenate. All samples used for bioassay titrations and Western
blotting were subjected to not more than one freezing and
thawing process to avoid distortion of results by loss of PrPres or
infectivity due to freezing.
Proteinase K Digestion, Western Blotting, and Quantification of
PrPres. For PrPres quantification, samples were thawed, digested
with proteinase K (100 ug/ml) for 1 h at 37°C, and analyzed by
Western blotting with 3F4-antibody at a 1:2,000 dilution (13).
PrP was visualized by enhanced chemiluminescence reaction
(Amersham Pharmacia, Freiburg, Germany) according to the
instructions of the manufacturer and was immediately evaluated
densitometrically by using the Diana III luminescence imaging
system (Raytest, Straubenhardt, Germany) and the AIDA software
package (Raytest). Protein sizes were determined by using
Bio-Rad high molecular weight marker.
Amplification Factors/Normalization. Amplification (Amp) factors
(AmpF) were determined in serial dilution experiments by
comparing the intensities (I) of diglycosylated PrP bands in the
molecular mass range between 27 and 30 kDa after amplification
to the corresponding PrP signal after the previous amplification
round (e.g., AmpF(II)  IafterAmpIIIafterAmpI  dilution factor),
except for cycle I where the AmpF was determined directly from
Amp(I)  IafterAmpIIbeforeAmpI as was completed for amplification
experiments without serial dilution.
In inhibition experiments with rPrP, all PrPres signals were
normalized to the amplification product obtained without the
addition of rPrP for each blot. Normalized mean values and SD
of 46 independent experiments are shown in each column (Fig.
2). P values were calculated by using Students paired t test
between the data sets of two samples as indicated. P values0.05
were considered to indicate statistically significant differences.
Bioassay for Titration of Infectivity. Archived samples were thawed
and diluted 1:1,000 in PBS, and 50-l aliquots were inoculated
i.c. into SHas as described (14). The amount of infectivity [50%
i.c. lethal doses (LD50i.c.)] in the inoculated samples (50 l) was
assayed as described by Kimberlin and Walker (14) by the
observed incubation times (t, in days) until terminal scrapie,
using doseincubation curves (15). For the calculation of infectivity
from incubation times, the following empirical equation
was used: log(LD50i.c.)  0.0008 t2  0.2575t  20.7929 [mean
error of assay:  0.4 log(LD50i.c.)]. For the purpose of fitting
logarithmic titers, infectivity in animals without scrapie symptoms
at 250 days after inoculation was set to 0.1 LD50i.c. and the
percentage of SHas was plotted against log(LD50i.c.).
Results and Discussion
Substrate-Dependent Generation of Protease-Resistant PrP. Analogous
to the approach used by Saborio et al. (4), we spiked brain
homogenate from normal SHas with brain homogenate from
terminally ill scrapie hamsters infected with strain 263K and
subjected them to sonication and incubation cycles at different
dilutions. The samples were then digested by proteinase K. The
amount of PrPres was quantified by Western blotting, using 3F4
antibody. Densitometric quantification of Western blot bands
showed a linear response to the amount of PrPres present in the
concentration range used in PMCA experiments. Sonicating
samples before loading onto the gel did not change the signal
(see Fig. 5, which is published as supporting information on the
PNAS web site). Variations in densitometric signal of parallel
samples were found to be 1020% between Western blots and
10% within the same blot.
Fig. 1a shows an increase in PrPres signals after amplification
by a factor of 5 (10 rounds) and 10 (23 rounds), respectively. No
significant increase in PrPres signals was observed after incubation
(23 h at 37°C) without sonication.
Next, we tested whether the observed increase in immunoreactive
PrP is due to generation of new PrPres. Alternatively, it
would be conceivable that PrPSc trapped in cellular membrane
fragments, which has not been detected originally, could have
been released by repeated sonication of the sample leading to an
apparent increase in PrPres. To address this concern, scrapie
hamster brain homogenate (263K) was diluted in brain homogenate
of PrP0/0 mice (11) lacking PrPC for the conversion
reaction. In this reaction mixture, no increase of the immunostaining
of hamster PrPres occurred (Fig. 1c). However, similarly
treated 263K brain homogenates diluted in brain homogenate of
healthy SHa showed an increase in PrPres immunostaining of
3.2- to 3.4-fold after eight PMCA rounds (Fig. 1b). Therefore,
the increased intensity of the Western blot signals for hamster
PrPres observed after PMCA in Fig. 1a cannot be simply
Fig. 1. (a) Amplification of PrPres. Brain homogenate (10% wtvol) prepared
from 263K-infected hamsters in the terminal stage of scrapie
(263Khomogenate)
was diluted 1:10, 1:100 and 1:200 in brain homogenate (10%
wtvol) from healthy SHas. Brain homogenates were subjected to 10 (lanes
58) and 23 (lanes 912) amplification cycles, and control samples were
frozen
immediately (lanes 14) or were incubated for 23 h at 37°C without
sonication
(lanes 1416). Samples were analyzed by Western blotting, using 3F4 antibody
after digestion with proteinase K. The amount of PrPres increased at least
10-fold after 23 amplification cycles (lane 1 vs. lane 8). (b and c)
Substratedependent
amplification. 263K homogenate was diluted 1:100 into brain
homogenate of healthy SHas (b) or PrP0/0 knockout mice (c). A 10-fold excess
of SHa rPrP (23231) (Prionics) was added where indicated and the reaction
mixture was subjected to eight PMCA amplification rounds. All experiments
were performed in duplicate and the mean deviation between two independent
samples after amplification was found to be30%. Samples subjected to
PMCA in hamster brain homogenate (b, lanes 5 and 6) showed an increase in
PrPres (3.2- to 3.4-fold), when compared with control samples (b, lanes
1 and
2), and amplification of PrPres could be blocked by excess SHa rPrP (b,
lanes 7
and 8). In brain homogenates devoid of PrPC, no amplification occurred with
or without the presence of hamster rPrP (c, lanes 18).
12208  www.pnas.orgcgidoi10.1073pnas.0404650101 Bieschke et al.
attributed to a release of previously undetected material but
must be due to PrPres newly generated from PrPC substrate.
Inhibition of PrPres Formation by rPrP. No detectable PrPres was
generated in PMCA samples containing SHa rPrP (23231) at a
10-fold excess to PrPC (Fig. 1b). To test whether the inhibition of
PrPres formation was specific, we added SHa rPrP at molar
rPrPPrPC ratios of 0.3:10 to the amplification reaction (Fig. 2).
Even substoichiometric quantities of SHa rPrP significantly inhibited
PrPres generation. Half-maximal PrPres formation occurred at
a molar ratio of 0.3 and SHa rPrP at a molar ratio of 1 almost
completely inhibited the conversion reaction (P  0.003). Murine
rPrP (23230), however, as well as albumin (BSA), did not significantly
decrease the amount of PrPres generated by PMCA, even
when added at a 10-fold excess over PrPC (Fig. 2).
These findings show that hamster rPrP (23231) inhibits the
PMCA conversion reaction specifically. Inhibition of PrP conversion
even by substoichiometric amounts of homologous rPrP
fits well into the model of prion replication. For example, the
inhibitor could stick to the active site in PrPSc, thus blocking
further PrPC conversion or compete with PrPC in binding a
cofactor necessary for conversion. A specific catalytic cofactor,
such as the proposed protein X (16), would be expected to have
different binding affinities to PrP from different species, making
it a likely target for the specific inhibition observed in PMCA.
Alternatively, Masel et al. (17, 18) have modeled in detail how
PrPC conversion is efficiently inhibited by PrP-substrate analogs,
which bind to the active center of conversion, e.g., the sticky ends
of PrPSc fibrils, without being converted properly into a catalytically
active conformation. The observed inhibition of amplification
indicates high specificity of the PMCA reaction at the
molecular level.
PMCA with Serially Passaged PrPres. To test for an autocatalytic
molecular conversion process, we generated PrPres by PMCA
and serially passaged the reaction mixture into fresh brain
homogenate.
In our experiment, each cycle of the amplification reaction
consisted of 10 rounds of alternating sonication and subsequent
incubation at 37°C. After 10 rounds, the reaction mixture was
passaged by diluting aliquots 1.5-, 2-, or 2.5-fold into fresh brain
homogenate from normal SHas or from PrP0/0 mice. By performing
nine sequential passages, the initial material was diluted by a factor
of 38, 512, or 3,815, respectively. Samples were taken before and
after the amplification reaction in each passage and analyzed as
above (Fig. 3 a and b and see Fig 6, which is published as supporting
information on the PNAS web site, for 1.5 and 2.5 dilutions).
The PrPres signal was 50% of the initial signal in the 2 dilution
series after 10 amplification cycles and 9 subsequent passages (Fig.
3c), which corresponds to an average amplification factor of 1.8 
0.3 per cycle. We found the amplification factor to be constant
during the passage series ranging from 1.5 to 2.0 (Fig. 3d). The
average amplification factor of all dilution experiments (1.8  0.4)
was independent of the dilution factor within experimental error,
with a total amplification factor of 270 and 280 for 2- and 2.5-fold
dilutions, respectively (Table 1).
In additional experiments with five serial passages and various
dilution factors, a higher apparent amplification factor was
sometimes observed during the initial round of amplification,
possibly resulting from a superimposed process of recruitment of
PrPSc from previously insoluble high molecular weight debris
(data not shown). However, in subsequent passages, we always
found amplification factors consistent with the results shown
above. Note also that amplification factors depended on the ratio
of PrPSc to PrPC, a 1:100 starting dilution of PrPSc, producing 4-
to 5-fold amplification after 10 rounds, whereas increasing PrPSc
concentration to 1:20 lowered the amplification factor to the
value observed in serial dilution experiments.
By serially amplifying PrPres and diluting it into fresh brain
homogenate, the reaction is not limited by the supply of PrPC
substrate. If the dilution factor exactly matches the growth rate,
the system, in principle, allows indefinite autocatalytic propagation
of an infectious agent (19). The observed slow decrease
in PrPres signal in the course of the amplificationdilution cycles
is consistent with an exponential growth process in which the
dilution factor slightly exceeds the growth factor.
If the initial PrPSc merely acted as a catalytic seed for the
formation of PrPres, however, amplification would only be
observed in the initial amplification cycles and the reaction
would quickly come to a halt as the original PrPSc is diluted out
exponentially. Likewise, a hypothetical mechanism that exposes
new catalytic surfaces in the initial PrPSc with each sonication
step would not result in maintained amplification, unless the
increase in exposed catalytic surfaces exactly matches the dilution
factor. Whereas an exponential increase in catalytic sites by
fragmenting the initial PrPSc is, in principle, possible over nine
dilution cycles, resulting in 512-fold smaller PrPSc particles,
fragmentation of PrPSc and thus amplification efficiency should
then increase monotonously with sonication time. In PMCA
reactions, however, the dependency of amplification efficiency
on sonication time shows a distinct maximum at a sonication of
520 s per round (data not shown).
Taken together, our findings outlined so far demonstrate that
PrPres does serve as a catalytically active template for structural
PrPC conversion in PMCA. This finding provides proof for autocatalytic
replication of misfolded PrPres in an ex vivo conversion
system. The PrPres catalyzes conversion of PrPC to PrPres apparently
as efficiently as observed for the PrPSc initially present in the
sample and furthermore exhibits a resistance to proteinase K
digestion indistinguishable from that of PrPSc. Therefore, at the
molecular level, PrPres formed in the PMCA reaction possesses the
key properties attributed to infectious PrPSc by the prion hypothesis,
i.e., proteinase K resistance and autocatalytic protein misfolding
activity.
Fig. 2. Inhibition of PrPres formation by rPrP during PMCA. Dilutions of
263K
homogenate in SHa-homogenate(1:100) containing a 0- to 10-fold excess of
SHa rPrP (23231) (Prionics) over PrPC were subjected to eight amplification
cycles. After proteinase K digestion and analytical Western blotting, PrPres
was quantified densitometrically. PrPres signals of 46 independent
experiments
were normalized by setting the densitometric signal after PMCA to a
relative intensity of one. Mean values and SD are shown. P values 0.05
indicate statistical significant differences between two mean
values.A10-fold
molar excess of mouse rPrP and BSA was given into additional samples as an
unspecific reference. Hamster rPrP significantly inhibits PrPres
amplification
when compared with amplification without SHa rPrP (**, P0.003) and when
compared with amplification in the presence of excess murine rPrP or BSA (*,
P  0.05).
Bieschke et al. PNAS  August 17, 2004  vol. 101  no. 33  12209
BIOPHYSICS
Infectivity of Material Generated by PMCA with Serially Passaged
PrPres. The catalytic conversion activity of PrPres created in the
serial PMCA reaction suggested that, according to the prion
model, it might exhibit biological infectivity in an appropriate
bioassay. To test for a gain in infectivity during serial PMCA, we
archived samples of the initial infectious brain homogenate
before amplification (sample A), of the PMCA reaction mixture
after 10 serial passages (sample B), and of the starting material
after a 29-fold dilution without PMCA (sample C) at 80°C. Of
each of the samples, 50-l aliquots were inoculated i.c. into 10
hamsters after a 103-fold dilution in PBS. The resulting incubation
times until the occurrence of terminal scrapie are shown in
Fig. 4a. With 107  11 days, sample A showed the shortest
incubation time, corresponding to a mean infectious titer in the
diluted sample of 250 LD50i.c. Only 8 of 10 hamsters challenged
with sample C developed disease until 250 days after inoculation,
with a mean incubation time of 138 days for the diseased animals.
If the inocula administered to the yet surviving animal in this
group contained no infectivity, the mean titer for sample C
would be 2 LD50i.c per 50 l. The mean incubation time for
sample B was 125  6 days, corresponding to 13 LD50i.c. in 50 l
of the diluted sample, and lay between the two other groups.
Apparent infectivity titers in individual animals showed a
narrower distribution in the PMCA sample B than in control
samples A and C, suggesting a more homogeneous distribution
of the infectious agent, which is most plausibly due to the
sonication process repeatedly performed during PMCA (Fig.
4b). Up to 10-fold increased apparent infectivity values generated
by ultrasonication of purified PrPSc in the presence of
liposomes have been reported (20, 21) and predicted by kinetic
modeling (22). However, control experiments comparing infectivity
titers in sonicated and untreated samples by using limited
dilution series of Rocky Mountain Laboratory mouse scrapie
strain (RML) mouse prions in PrP-overexpressing mice (Tga20)
showed no significant difference in titers (data not shown).
Thus, the titration experiments do not provide conclusive evidence
whether PrPres formed by PMCA is associated with new
infectivity because the moderate increase in infectivity observed
Fig. 3. PMCA with serially passaged PrPres. (a) 263K-homogenate was
diluted 1:20 in SHa-homogenate for PMCA. One cycle consisting of 10
rounds of
amplification was performed and the reaction mixture was diluted 2-fold
in normal hamster brain homogenate after the cycle. The process was
repeated to a
total of 10 amplificationdilution cycles. Samples of 20 l were taken
before and after each cycle, frozen for storage, digested with
proteinase K, and subjected
to Western blot analysis by using 3F4 antibody and quantified
densitometrically. Lane X (1:200): a 10-fold dilution of the starting
homogenate was included as
a PrP concentration reference. (b) Sonicationdilution cycles were
carried out as in a by diluting into brain homogenate (10% wtvol) from
PrP0/0 mice. Uninfected
hamster brain homogenate was treated with proteinase K under identical
conditions to the PMCA samples to document complete proteinase K (PK)
digestion
of PrPC (lanesPK andPK). (c) Quantification of PrPres in the serial
transmission PMCA experiment shown in a; relative amounts of initial
PrPSc (filled bars) and
newly formed PrPres (open bars). (d) Amplification factors derived from
c. The catalytic activity is independent of the ratio of PrPSc derived
from the initial scrapie
homogenate and newly formed PrPres. The average amplification factor was
1.75  0.3 with a 270-fold total amplification. Serial transmission PMCA was
repeated several times for 510 consecutive cycles with different
dilution factors (data not shown), yielding 20% variation in
amplification factors.
Table 1. Amplification factors in serial transmission PMCA
Dilution factor per cycle 1.5 2.0 2.5
Amplification per cycle 1.7  0.4 1.75  0.3 1.8  0.3
Total amplification factor 50* 270 280
*Eight amplification cycles.
12210  www.pnas.orgcgidoi10.1073pnas.0404650101 Bieschke et al.
might well be due to fragmentation of PrPSc. Clearly, the rate of
PrPres generation is not matched by a corresponding increase of
infectivity during the PMCA reaction. Possibly, this specific form
of misfolded PrP needs additional structural features for infectivity
or it might require a specific cofactor that may not be incorporated
into the prion during PMCA such as highly complex polysaccharides,
which have been found associated with purified PrPSc (23).
Short RNA molecules have been implicated as essential components
of the in vitro amplification of PrPres and thus possibly for
infectivity (9). A drop in amplification efficiency is observed in the
last reaction cycle (Fig. 3d). Even though preparation of brain
homogenates and sonication of PMCA samples were carried out
under sterile conditions, 100 h of incubation at 37°Cmight still have
led to microbial growth and elevated proteolytic or RNase activity,
destroying potential cofactors for prion infectivity.
Conclusion
We serially passaged misfolded protease-resistant PrPres in an in
vitro amplification reaction. Amplification depends on cellular
PrPC as a substrate for conversion. Hamster rPrP acts as an
inhibitory substrate analogue, which specifically blocks PrPC
conversion at stoichiometric concentrations. Therefore, the
PMCA assay promises to be a valuable tool to screen compounds
for their therapeutic and prophylactic potential in fighting
transmissible spongiform encephalopathies.
PrPres generated by serial in vitro PMCA is indistinguishable
from PrPSc in terms of proteinase K resistance and catalyzes
PrP misfolding as efficiently as PrPSc. However, if PrPres
generated by the PMCA reaction is infective, its titer is at least
10-fold lower than PrPSc derived from the brains of Scrapie
hamsters, which suggests that the final make-up of PrPSc in
terms of conformation and aggregation state or an essential
cofactor for efficient infection of animals is lacking from the
PrPres produced in vitro.
On the other hand, serially passaging misfolded PrPres in the
PMCA procedure has produced misfolded PrP in an in vitro
reaction that meets all of the requirements of the biochemical PrP
misfolding process as postulated by the prion hypothesis, providing
formal proof for the autocatalytic propagation of PrP misfolding.
We thank U. Bertsch (Center of Neuropathology and Prion Research,
Ludwig Maximilians University of Munich) for rPrP, and Marion Joncic
(Robert Koch-Institute) and Salah Soliman (Center of Neuropathology and
Prion Research, Ludwig Maximilians University of Munich) for technical
assistance. This work was supported by European Union Grant QLK3-CT-
2001-02345, State of Bavaria Grants 830302-6 and 822796-9, and Ludwig
Maximilians University of Munich Fo¨FoLe Grant 822784-5.
1. Prusiner, S. B. (1982) Science 216, 136144.
2. Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. USA 95, 1336313383.
3. Kocisko, D. A., Priola, S. A., Raymond, G. J., Chesebro, B.,
Lansbury, P. T.,
Jr., & Caughey, B. (1994) Nature 370, 471474.
4. Saborio, G. P., Permanne, B. & Soto, C. (2001) Nature 411, 810813.
5. Jarrett, J. T. & Lansbury, P. T., Jr. (1993) Cell 73, 10551058.
6. Eigen, M. (1996) Biophys. Chem. 63, A1A18.
7. Lucassen, R., Nishina, K. & Supattapone, S. (2003) Biochemistry 42,
41274135.
8. Vorberg, I. & Priola, S. A. (2002) J. Biol. Chem. 277, 3677536781.
9. Deleault, N. R., Lucassen, R. W.&Supattapone, S. (2003) Nature 425,
717720.
10. Zobeley, E., Flechsig, E., Cozzio, A., Enari, M. & Weissmann, C.
(1999) Mol.
Med. 5, 242243.
11. Bu¨eler, H., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H. P.,
DeArmond,
S. J., Prusiner, S. B., Aguet, M. & Weissmann, C. (1992) Nature 356,
577582.
12. Liemann, S. & Glockshuber, R. (1999) Biochemistry 38, 32583267.
13. Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M.,
Fersko, R.,
Carp, R. I., Wisniewski, H. M. & Diringer, H. (1987) J. Virol. 61,
36883693.
14. Kimberlin, R. H. & Walker, C. A. (1986) J. Gen. Virol. 67, 255263.
15. Kimberlin, R. H. & Walker, C. A. (1977) J. Gen. Virol. 34, 295304.
16. Prusiner, S. B., Scott, M. R., DeArmond, S. J. & Cohen, F.E. (1998)
Cell 93, 337348.
17. Masel, J. & Jansen, V. A. (2000) Biophys. Chem. 88, 4759.
18. Masel, J., Jansen, V. A. & Nowak, M. A. (1999) Biophys. Chem. 77,
139152.
19. Spiegelman, S., Haruna, I., Holland, I. B., Beaudreau, G. & Mills,
D. (1965)
Proc. Natl. Acad. Sci. USA 54, 919927.
20. Gabizon, R., McKinley, M. P. & Prusiner, S. P. (1987) Proc. Natl.
Acad. Sci.
USA 84, 40174021.
21. Gabizon, R. & Prusiner, S. B. (1990) Biochem. J. 266, 114.
22. Masel, J. & Jansen, V. A. (1999) Proc. R. Soc. London Ser. B 266,
19271931.
23. Appel, T. R., Dumpitak, C., Matthiesen, U. & Riesner, D. (1999) Biol
Chem.
380, 12951306.
Fig. 4. Incubation timesandcorresponding infectivity titers in hamster
bioassay
experiments. The following samples were analyzed: sample A, initial reaction
mixture taken beforePMCAand containing 5103 g of scrapie brain tissue per
ml (); sample B, PMCA reaction mixture after 10 serial passages (F);
and sample
C, initial reaction mixture after 512-fold dilution without PMCA ().
Samples B
and C each contained 1105 g of the initially added scrapie brain
tissue per ml.
The 50-l aliquots of the samples were inoculated i.c. into 10 SHas
after 103-fold
dilution in PBS. (a) Mean incubation times were 107  11 and 125  6
days for
samples A and B, respectively, before and after serial PMCA. Only 8 of
10 animals
challengedwithsampleCsuccumbedto scrapie until250days after infectionwith
a mean incubation time of 138 days for the diseased animals. (b)
Infectivity titers
werecalculatedfromincubation times by doseincubation curves as in ref.
15and
were plotted for individual animals on a logarithmic scale. The
distribution of
titers could be described by a linear fit with a slope of 30  3 for
samples A and
Candameanlogarithmic titer of 2.4 (250LD50i.c.)and0.5 (3LD50i.c.),
respectively.
The infectivity distribution of sample B showed a slope of 73  5 with a
mean
logarithmic titer of 1.1 (13 LD50i.c.). Titers refer to the mean
infectivity in 50-l
inocula of samples AC.
Bieschke et al. PNAS  August 17, 2004  vol. 101  no. 33  12211
BIOPHYSICS

TSS

######### http://mailhost-alt.rz.uni-karlsruhe.de/warc/bse-l.html ##########





Follow Ups:



Post a Followup

Name:
E-mail: (optional)
Subject:

Comments:

Optional Link URL:
Link Title:
Optional Image URL: