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J. Biol. Chem., Vol. 276, Issue 34, 31479-31482, August 24, 2001
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From the Department of Neurology, the Agnes Ginges Center for Human
Neurogenetics, Hadassah University Hospital,
Jerusalem 91120, Israel
Received for publication, May 28, 2001, and in revised form, June 14, 2001
Prion protein (PrP)Sc,
the only known component of the prion, is present mostly in the brains
of animals and humans affected with prion diseases. We now show that a
protease-resistant PrP isoform can also be detected in the urine of
hamsters, cattle, and humans suffering from transmissible spongiform
encephalopathies. Most important, this PrP isoform
(UPrPSc) was also found in the urine of hamsters inoculated
with prions long before the appearance of clinical signs.
Interestingly, intracerebrally inoculation of hamsters with
UPrPSc did not cause clinical signs of prion disease even
after 270 days, suggesting it differs in its pathogenic properties from brain PrPSc. We propose that the detection of
UPrPSc can be used to diagnose humans and animals
incubating prion diseases, as well as to increase our understanding on
the metabolism of PrPSc in vivo.
Since the appearance of
BSE1 in 1985 (1, 2) the need
for an in vivo diagnostic test for prion diseases has become
acute. In the absence of such a method, an extensive slaughtering of cattle was required once an affected animal was identified within a
herd. The need for such an in vivo test was reinforced since the first cases of variant Creutzfeldt-Jakob disease were reported in
1996 (3-5). Variant Creutzfeldt-Jakob disease is a fatal
neurodegenerative disease believed to be caused by the consumption of
BSE-contaminated meat, and the incubation time between infection to
clinical symptoms may be as long as decades (4). As opposed to cattle,
the incubating individuals (which at this point can be any of us), will
be present for many years, donating blood, and in some cases other
organs, to the non-affected population.
The only identified component of the prion, the agent causing prion
diseases (also referred to as transmissible spongiform encephalopathies; TSEs), is PrPSc, a protease-resistant
abnormal isoform of PrPC. PrPC is a
glycophosphatidyl inositol-anchored glycoprotein of unknown function (7, 8). Although some other markers for prion diseases have
been suggested (9-11), PrPSc remains not only an
obligatory prion component but also the only reliable and universally
accepted marker for this family of diseases (12-14). We now show that
a protease-resistant isoform of PrP, hereby denominated
UPrPSc, can be detected, following a specific enrichment
procedure, in the urine of scrapie-infected hamsters, BSE-infected
cattle, and humans suffering from CJD. These results pave the way for the development of a simple in vivo test for prion diseases.
Analysis of Urine Samples--
Urine samples (2 ml for hamster,
10 ml for human, and 15 ml for bovine) were sedimented for 5 min at
3000 rpm to discard occasional cell debris and then dialyzed overnight
in a cellulose tubular membrane (pore range 6000-8000 dalton;
Membrane Filtration Products, Inc., Seguin, TX) against 5 liters
of saline at 4 °C (saline was changed twice during dialysis). For
experimental purposes, (see Fig. 1c), the dialysis step was
omitted in some cases. Subsequently, the urine samples were centrifuged
at high speed (100000 gav (average gravitational
force equivalent executed by the centrifugation process on the sample)
for 1 h at 4 °C). Pellets were resuspended in 100 µl of 2%
sarkosyl STE buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA). Samples were divided and
digested in the presence or absence of proteinase K (PK). Digestion
conditions were optimized for each species. For hamster urine, 40 µg/ml PK for 60 min at 37 °C; for human urine, 40 µg/ml PK for
30 min at 37 °C; for bovine urine, 20 µg/ml for 30 min at
37 °C.
Following protease digestion, the urine samples were boiled in SDS
sample buffer, applied to a 12% SDS-polyacrylamide gel electrophoresis
and subsequently transferred to a nitrocellulose membrane. Membranes
were blocked with 3% fat milk except for the bovine samples, which
were blocked with 5% human serum albumin; Sigma. A second blocking
step was performed with a mixture of 1:3000 anti-mouse IgG and 1:3000
anti-rabbit IgG in TBST buffer (10 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0.05% Tween 20; for 30 min) to avoid
nonspecific binding of the secondary antibody to IgG light chain
present in some urine samples. Membranes were then rinsed in TBST for
15 min and immunoblotted with either In Vivo Experiments--
Syrian hamsters were inoculated with
samples containing urine PrP from normal or scrapie-sick hamsters. For
inoculation, urine samples were prepared as described above (including
PK digestion but not SDS boiling) and diluted as required in 1% bovine
serum albumin/phosphate-buffered saline. Brain samples from
scrapie-infected hamsters were diluted to contain similar
concentrations of PrPSc (see Fig. 3) and inoculated to
additional groups of hamsters. To achieve similar concentrations of
protease-resistant PrP in brain and urine inoculi, each animal was
inoculated, depending on the appropriate experimental group, with 50 µl of sample containing PrP originating from either 0.5 ml of urine
or from 1.25 µl of 10% scrapie hamster brain homogenate.
Collection of Urine from Hamsters--
Following inoculation,
animals were examined daily for scrapie-associated symptoms. For time
course experiments, groups of three hamsters in an equivalent stage of
disease incubation were housed each week in a metabolic cage for urine
collection from 15:00 to 08:00 of the next day. Urine was collected in
the morning and immediately frozen at Tissue Homogenates--
Whole brain or kidney were homogenized
in 10 volumes of homogenization buffer (10 mM Tris-HCl, pH
7.5, 300 mM sucrose/phosphate-buffered saline). Following
centrifugation (2000 rpm, 15 min, 4 °C), the supernatant was frozen
( Human Urine Samples--
Whenever possible, the human samples
from CJD patients and controls were the first morning urine. Some CJD
and post-stroke patients were bearing catheters, and in these cases
urine was collected for a period of up to 8 h in a urine
collecting bag. All samples were frozen until further use.
Bovine Urine Samples--
All BSE and most control bovine urine
samples were obtained from the Veterinary Laboratory Agency (VLA) in
London, United Kingdom. The VLA samples constituted 51 samples of 24 cows, all coded for blind testing. Additional freshly frozen control
samples were obtained from the Hebrew University Veterinary School.
According to VLA records, most samples were frozen following collection whereas some were kept chilled. No information was provided regarding time of day for sample collection.
Most of the CJD patients tested in this work (6 of 8) were genetic
patients carrying the E200K mutation (15-18). One of the patients was
a 52-year-old individual homozygous for this mutation (19). Among the
other genetic patients, four were MM at codon 129 and one was
MV. The E200K mutation is located at a Methionine 129 allele (17). The
human controls (n = 15), were either healthy individuals (n = 7) or patients suffering from diverse
neurological disorders, such as Alzheimer's disease (n = 3), multiple sclerosis (n = 2), and stroke
(n = 3). Urine from BSE-infected cattle, as well as
most of the bovine controls used in this work, were obtained from the
VLA laboratory in the United Kingdom. For the bovine samples, the urine
test was performed as a blind study. Urine from Syrian hamsters,
inoculated with either the 263K prion strain (20) or normal
controls, were collected with the use of a metabolic cage (as explained
under "Experimental Procedures"), as pools from three animals.
Urine samples from scrapie-infected hamsters, CJD patients, and
BSE-infected cattle, as well as from their appropriate controls, were
processed for enrichment of UPrPSc as described under
"Experimental Procedures" and subsequently immunoblotted for PrP
peptides. Human and hamster urine samples were immunoblotted with
either mAb 3F4 or 6H4 (not shown), whereas bovine samples were blotted
only with mAb 6H4. Parallel samples were blotted only with secondary
Fig. 1 demonstrates the results of such
an experiment. Although a precipitable and protease-resistant form of
PrP could be detected in the dialyzed urine of prion disease-affected
humans and animals, this was not the case for the urine of the
appropriate controls. The PrP urine assay was negative for all controls
humans (n = 15), hamsters (n = 10),
when each sample represents a pool from three hamsters, and cattle
(n = 15), positive for all CJD patients tested
(n = 8), for a large number of hamster groups (n = 20), and for 10 of 12 BSE-infected cattle, whereas
the other two BSE-positive cows showed a positive but poor signal. As
can be seen in Fig. 1c dialysis was essential for the
detection of the protease-resistant UPrPSc in urine from
scrapie-infected hamsters. The fact that the PrP signal in urine could
be blocked by the 3F4 peptide and did not react with the secondary
antibody provides strong evidence that this signal belongs to a PrP
peptide (Fig. 1d)
A surprising result depicted above is that a protease-sensitive PrP
isoform is present in the precipitable fraction of the normal urine
samples, as opposed to what is expected for PrPC. It is to
be noticed, however, that no detergent was added to the urine before
ultracentrifugation as performed in membrane extractions that result in
a soluble PrPC (21, 22). It is also possible that all PrP
molecules are present in urine in a partially denatured state because
of the presence of urea. Also dialysis of normal urine may induce the aggregation of the PrPC isoform, which, as opposed to
UPrPSc, is protease-sensitive. Although the exact chemical
nature of UPrPSc is yet to be determined, its molecular
weight seems to be slightly higher than full-length and fully
glycosylated PrPC or PrPSc. In addition, the
pattern of UPrPSc in the immunoblots suggest it may be
composed mostly of the higher molecular band of PrP and not of the less
glycosylated species. This may indicate that partially or non
glycosylated PrP is less resistant to the conditions encountered by
PrPSc until it is excreted in urine as UPrPSc.
That normal PrP is excreted in urine is not entirely surprising, because this is also the case of other GPI-anchored proteins
(23-25).
UPrPSc did not originate directly from the kidneys, because
no PrPSc could be identified in the kidney tissue of the
scrapie-infected hamsters (Fig. 1b). This suggests
UPrPSc originates from other organs and arrives to the
urine from blood.
The detection of PrPSc at the end stages of prion disease
may result from some degree of blood brain-barrier disruption by brain degeneration (26). Contrarily, the presence of PrPSc in
prion-infected urine early in the incubation time would suggest a
clearance pathway for the aberrant PrP protein either from brain or
from a peripheral organ, through its excretion into urine. To address
this question, we inoculated Syrian hamsters either intracerebrally
(i.c.) or intraperitoneally (i.p.) with hamster prions and collected
urine samples, as described under "Experimental Procedures," every
week during the incubation period. Each sample was frozen immediately
after collection. At the end of the experiment, similar volumes of
these urine samples were thawed, enriched for PK-resistant
UPrPSc as described above, and subsequently immunoblotted
with The results of such an experiment can be seen in Fig.
2. A light signal of prion-specific PrP
was detected in the urine samples of the i.c. inoculated hamsters after
only 17 days (Fig. 2a), following by the disappearance of
the PrP signal until day 35. Subsequently, the signal for
protease-resistant PrP increased until the appearance of clinical
signs. Similar results were obtained for the i.p. inoculated hamsters.
A PrP signal was detected in the first weeks following the inoculation,
disappeared at later dates, and reappeared at about 60 days. These
results may infer that some of the prion inoculum is immediately
secreted following inoculation. Thereafter, no PrP signal appeared in
urine until the first stages of prion protein accumulation in brain.
Indeed, whereas scrapie incubation time for i.c. or i.p. inoculated
hamsters with the 263K strain is about 75 and 120 days, respectively,
PrPSc can be identified in enriched brain samples of these
hamsters at about 40 (i.c.) or 70 (i.p.) days (27-29). Our experiments
therefore suggest that UPrPSc is excreted in urine in
parallel to its accumulation in brain.
ACCELERATED PUBLICATION
A Protease-resistant Prion Protein Isoform Is Present in
Urine of Animals and Humans Affected with Prion Diseases*
ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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PrP mAb 3F4 or 6H4 (hamster
and human) at 1:5000 or 6H4 (bovine) at 1:5000.
80 °C. Food and water were
supplied ad libitum. A similar procedure was applied to
scrapie-sick hamsters.
80 °C).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mouse antisera and showed no interfering signals.
View larger version (47K):
[in a new window]
Fig. 1.
Protease-resistant PrP in urine of
TSE-affected humans and animals. a, freshly frozen
urine samples from hamsters, humans, and cattle were enriched for
protease-resistant PrP as described under "Experimental
Procedures." All samples were digested in the presence or absence of
proteinase K, as described under "Experimental Procedures," and
immunoblotted with either PrP mAb 3F4 (hamster and human samples) or
6H4 (bovine samples). 1, homozygous E200K CJD patient;
2, heterozygous E200K CJD patient; 3, human
control; 4, scrapie-sick hamster; 5, normal
hamster; 6, BSE-sick cattle; 7, normal cattle.
The volumes of urine used are described under "Experimental
Procedures." b, 5 µl of a 10% brain samples were
analyzed for PrP. 1, homozygous CJD patient; 2,
heterozygous CJD patient; 3, human control; 4,
scrapie-sick hamster; 5, normal hamster; 6,
kidney sample from scrapie-sick hamster. c, scrapie hamster
urine samples were enriched for UPrPSc with and without the
dialysis step. Samples were digested in the presence or absence of PK
as described under "Experimental Procedures." d, human
brain sample (b) and human urine sample (U) were
immunoblotted with mAb 3F4 in the absence (1) or the
presence (2) of 10 µg/ml of the peptide comprising the 3F4
epitope. Molecular mass markers (top to
bottom) were 36 and 30 kDa.
PrP mAb 3F4.
View larger version (38K):
[in a new window]
Fig. 2.
Prion-specific PrP can be detected during
scrapie incubation time. Urine samples were collected weekly from
Syrian hamsters inoculated either i.c. (a) or i.p.
(b) with hamster 263K prions and enriched for
UPrPSc as described. Samples were immunoblotted with PrP
mAb 3F4. The arrows represent the onset of clinical signs.
The molecular mass marker was 30 kDa.
The results of the experiments described in both Figs. 1 and 2 indicate therefore that urine testing for protease-resistant PrP can be used not only to diagnose prion diseases in animals and humans at terminal stages of the disease but also to diagnose these diseases in the subclinical stages of infection. If indeed the PrP signal detected at the first weeks-post infection is because of clearance of the inoculum, the PrP urine test may serve to diagnose a potential new occurrence of infection. This will allow in the future, providing an effective anti-prion therapy becomes available, to treat individuals at risk of a new prion exposure.
The detection of UPrPSc raises the alarming possibility that urine from prion-infected individuals, either ill or as yet incubating the disease can somehow transmit prion diseases. This prospect is especially disturbing in the case of BSE-infected cattle, as well as in natural scrapie in sheep. Because the mechanism by which these diseases are transmitted among animals within the herd was never elucidated (30, 31), it is conceivable that urine can contaminate the living areas of these animals.
To investigate whether urine from TSE-infected animals can be
infectious, we inoculated 20 hamsters with UPrPSc pooled
and enriched from urine of 10 hamsters terminally ill with scrapie. 20 hamsters were inoculated with similarly prepared samples from 10 normal
hamsters. Brain samples from scrapie-infected hamsters, which were
diluted to contain similar concentrations of PrPSc (1.25 µl of 10% homogenate) to the enriched UPrPSc (from 0.5 ml of urine), were inoculated to additional groups of hamsters (Fig.
3a). Hamsters were observed
daily for symptoms of scrapie infection. Urine was collected
periodically from animals inoculated with UPrPSc. At
different time points during the experiment, some of the hamsters
inoculated with UPrPSc were sacrificed and tested for the
presence of PrPSc in their brains.
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As expected, the animals inoculated with scrapie-infected brain samples
suffered from fatal disease symptoms at about 80 days post-inoculation.
Contrarily, none of the animals inoculated with urine samples (normal
or scrapie-infected) developed clinical symptoms of prion disease to
date (270 days post-infection). 12 hamsters (four groups of three),
inoculated with scrapie urine samples, were tested for the presence of
UPrPSc, and all were found positive from about 60 days
post-inoculation (Fig. 3, b, 2). In addition, low
concentrations of PrPSc could be identified in the brain of
one of three hamsters sacrificed at about 120 days (Fig. 3,
b, 3). All other hamsters in this experiment are
still under observation to determine whether they will develop a fatal
prion disease at a later date. These results suggest that UPrPSc inoculation can result in a subclinical or carrier
state prion infection. The clinical and epidemiological implications of
this finding are yet to be determined.
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Why is UPrPSc excreted into urine? Because most urine proteins originate from blood, we speculate that some PrPSc, either from brain or from a peripheral organ, is released during the disease incubation into the blood serum in a non-aggregated form, although at a low and undetectable concentrations. Because of its protease resistance, this PrPSc is not digested by blood proteases. However, and because the MW of PrP is below the cut off size for filtering through kidney cells (about 40 kDa) (32), PrP may subsequently be secreted into the urine and thereby be concentrated, as other proteins, at about 120 times over its concentration in blood (32). The concentration by the kidney makes it possible to detect PrPSc in urine much easier than in blood. Because dialysis of the urine seems to be a necessary step in our detection procedure, we propose that UPrPSc is present in a semi-denatured form, probably because of the relative high concentrations of urine-denaturing agents, and is subsequently renatured during the dialysis step. This denaturation/renaturation effect may also happen in the field because of absorption of the urea by the soil.
UPrPSc may differ in its conformation from brain PrPSc, thereby explaining the fact that inoculation of comparable amounts of both protease-resistant PrP isoforms produced such different results. It is to be remembered however that not all protease-resistant PrP molecules carry prion infectivity. Indeed, in vitro conversion experiments of PrPC to PrPSc, in which protease resistance was achieved by a denaturation/renaturation procedure, resulted in a protease-resistant but not infectious PrP isoform (33, 34). Contrarily, UPrPSc may resemble the new protease-resistant soluble isoform we have identified lately, which is associated with very low levels of infectivity, if any (35, 36). The latest possibility is consistent with the fact that UPrPSc is found in urine, because an aggregated molecule could not filter through the kidney.
It can also be speculated that UPrPSc is a component of a new prion strain, less virulent than the original 263K strain, which may produce not a fatal but a subclinical or a carrier state prion disease. Recent publications indicate the presence of low levels of PrPSc in the brains of animals that did not succumb to prion disease (6, 37), suggesting a subclinical state of prion disease may exist. In some cases, the brains of these animals transmitted a fatal prion disease to other rodents, suggesting apparently healthy carriers of prions disease can transmit disease.
To summarize, we have identified a prion-specific protease-resistant
PrP isoform in the urine of prion-infected animals and humans
(UPrPSc) that may be used for the in vivo early
diagnosis of ill, as well as seemingly healthy but prion-infected,
individuals. Our findings, in addition to their practical aspects, may
also open new avenues to investigate the metabolism and clearance
mechanism of PrPSc during prion infection and disease.
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ACKNOWLEDGEMENTS |
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We thank Prof. Abramsky from the Hadassah Hospital Neurology Department and Prof. Kahana from the Barzilai Hospital Neurological Unit, as well as the entire neurological community in Israel for collaboration in providing samples from CJD patients and useful discussions. We are very grateful to the VLA laboratory in the United Kingdom for providing urine from normal and BSE-infected cattle, as well as the Hebrew University Veterinary School for providing normal bovine urine.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 972-2-6429441;
E-mail: gabizonr@hadassah.org.il.
Published, JBC Papers in Press, June 21, 2001, DOI 10.1074/jbc.C100278200
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ABBREVIATIONS |
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The abbreviations used are: BSE, bovine spongiform encephalopathy; TSE, transmissible spongiform encephalopathy; PrP, prion protein; CJD, Creutzfeldt-Jakob disease; PK, proteinase K; mAb, monoclonal antibody; VLA, Veterinary Laboratory Agency; i.c., intracerebrally; i.p., intraperitoneally.
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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