Tissue plasminogen activator in brain tissues infected with transmissible spongiform encephalopathies.

Abstract

Prion propagation involves conversion of host PrP(C) to a disease-related isoform, PrP(Sc), which accumulates during disease and is the principal component of the transmissible agent. Proteolysis seems to play an important role in PrP metabolism. Plasminogen, a serine protease precursor, has been shown to interact with PrP(Sc). Plasminogen can be proteolytically activated by tissue plasminogen activator (tPA). Recent reports imply a crosstalk between tPA-mediated plasmin activation and PrP. In our study, both tPA activity and tPA gene expression were found elevated in TSE-infected brains as compared to their normal counterparts. Furthermore, it was proved that PrP(Sc), in contrast to PrP(C), could not be degraded by plasmin. In addition, it was observed that TSE symptoms and subsequent death of plasminogen-deficient and tPA-deficient scrapie challenged mice preceded that of wild-type controls. Our data imply that enhanced tPA activity observed in prion infected brains may reflect a neuro-protective response.

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Tissue plasminogen activator in brain tissues infected with
transmissible spongiform encephalopathies
K. Xanthopoulos,aI. Paspaltsis,aV. Apostolidou,aS. Petrakis,aC.J. Siao,cA. Kalpatsanidis,b
N. Grigoriadis,bA. Tsaftaris,dS.E. Tsirka,cand T. Sklaviadisa,d,*
aPrion Disease Research Group, Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences,
Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
bB’ Department of Neurology, School of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
cDepartment of Pharmacological Sciences, University Medical Center at Stony Brook, Stony Brook, NY 11794-8651, USA
dCentre for Research and Technology-Hellas, Institute of Agrobiotechnology, CERTH/INA 6th km Charilaou, Thermi, 57001 Thessaloniki, Greece
Received 23 September 2004; revised 5 April 2005; accepted 14 April 2005
Available online 26 May 2005
This article is dedicated to the memory of Professor Stelios Orphanoudakis.
Prion propagation involves conversion of host PrPCto a disease-related
isoform, PrPSc, which accumulates during disease and is the principal
component of the transmissible agent. Proteolysis seems to play an
important role in PrP metabolism. Plasminogen, a serine protease
precursor, has been shown to interact with PrPSc. Plasminogen can be
proteolytically activated by tissue plasminogen activator (tPA). Recent
reports imply a crosstalk between tPA-mediated plasmin activation and
PrP. In our study, both tPA activity and tPA gene expression were
found elevated in TSE-infected brains as compared to their normal
counterparts. Furthermore, it was proved that PrPSc, in contrast to
PrPC, could not be degraded by plasmin. In addition, it was observed
that TSE symptoms and subsequent death of plasminogen-deficient
and tPA-deficient scrapie challenged mice preceded that of wild-type
controls. Our data imply that enhanced tPA activity observed in prion
infected brains may reflect a neuro-protective response.
D 2005 Elsevier Inc. All rights reserved.
Keywords: tPA; Plasminogen; PrP; TSE
Introduction
Prions are proposed to be propagated through transmission of a
disease-related isoform of the prion protein (Prusiner, 1982). PrPC
is a glycoprotein, mainly located in neuronal tissue, but detectable
in other tissues as well (Manson et al., 1992; Moser et al., 1995).
The physiological role of prion protein has not been defined yet.
There are some indications it may act as a cellular receptor, as a
protein receptor (Martins et al., 1997) and as a protein involved in
cell signaling (Massimino et al., 2002). There is evidence about an
active role of prion protein in the metabolism of copper (Brown et
al., 1997; Pauly and Harris, 1998) and furthermore, it is suggested
that it has a protective role for the cell against oxidative stress
(Brown et al., 1999, 2001; Ellis et al., 2002; Klamt et al., 2001).
Human tPA is an approximately 68 kDa molecular weight serine
protease composed of 527–530 amino acids. It has two kringle
domains involved in its binding to fibrin and possibly in its binding
to a prion–plasminogen complex (Ryou et al., 2003). It is secreted
mainly from vascular endothelial cells and in the nervous system
fromneuronsandmicroglia(Tsirkaetal.,1997).Itcanbedetectedin
most biological fluids; its concentration in plasma is 50 Ag/l. tPA is
primary involved in the activation of the inactive zymogen
plasminogen to the active protease plasmin by hydrolyzing the
peptidicbondbetweenresiduesR561andV562.Plasminisinvolved
in a variety of biological processes, such as cell migration, growth,
inflammation and tumor invasion, but its primary function is to
dissolve blood clots in the vasculature. It has been reported that tPA
is secreted in response to mental stress (Jern et al., 1989) and that it
plays a role in neurodegeneration (Qian et al., 1993; Tsirka et al.,
1995, 1996, Yepes et al., 2000) and neuronal plasticity (Baranes et
al.,1998;Qianetal.,1993).ItwasrecentlyshownthattPAcancross
the blood–brain barrier via interaction with one of its cell surface
receptors, the LDL receptor-related protein (Yepes et al., 2003).
PlasminogenhasbeenreportedtohavehighaffinityforPrPScbut
not for PrPC(Fischer et al., 2000; Maissen et al., 2001). However,
morerecentstudiesprovedthatplasminogencanbeboundtohuman
recombinant PrP, which resembles the normal isoform of PrP (PrPC)
(Ellis et al., 2002; Kornblatt et al., 2003; Praus et al., 2003). The
thermodynamics and the kinetics of the complex formation between
0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2005.04.008
* Corresponding author. Laboratory of Pharmacology, Department of
Pharmaceutical Sciences, School of Health Sciences, Aristotle University of
Thessaloniki, 54124 Thessaloniki, Greece. Fax: +30 2310997645.
E-mail address: sklaviad@auth.gr (T. Sklaviadis).
Available online on ScienceDirect (www.sciencedirect.com).
www.elsevier.com/locate/ynbdi
Neurobiology of Disease 20 (2005) 519 – 527

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human recombinant PrP and plasminogen have been studied by
Cuccioloni et al. (2004), whereas Kornblatt et al. (2004) have
studied the pressure and thermal sensitivity of the complex between
recombinant sheep PrP and human plasminogen.
The biological role of this interaction has not yet been
elucidated, but given the complex formation, plasminogen has
been considered to be a possible carrier of PrPScto the central
nervous system, through the blood circulation (Fischer et al.,
2000). As far as plasmin-mediated PrP proteolysis is concerned, it
has been shown that soluble recombinant PrP is cleaved by plasmin
(Kornblatt et al., 2003; Praus et al., 2003) and that it can stimulate
tPA activity (Ellis et al., 2002; Praus et al., 2003). Ellis et al.
reported that the plasminogen activation system is stimulated by
the presence of apo-PrP, i.e. PrP not bound to Cu2+, whereas Praus
et al. found that the stimulation occurs through its NH2-terminal
fragment (PrP23–110) (Praus et al., 2003). The latter is in
accordance with an earlier finding that tPA-mediated plasminogen
activation can be stimulated by partially denatured proteins as well
(Machovich and Owen, 1997).
In light of these findings, we assessed tPA activity in TSE-
infected tissue and evaluated tPA gene expression. We also verified
PrPScstability towards plasmin-mediated proteolytic degradation
and challenged tPA and plasminogen-deficient mice with scrapie
agents. As expected, PrPSccontrary to PrPCproved to be protease-
resistant. Interestingly, tPA activity was found elevated, in accord-
ance to elevated tPA gene expression, while the tPA and the
plasminogen-deficient mice appeared to be more susceptible to
TSEs than their wild-type counterparts.
Materials and methods
Tissue homogenates and specific reagents
All the tissues in the study originated from animals of the same
age, gender and clinical stage, with similar living conditions.
Following sacrifice, tissues were stored at ?70-C, and subsequent
thawing was accomplished by placing them in an ice bucket for the
necessary amount of time. Hamster scrapie 263K strain was kindly
provided by Dr. R. Gabizon, Hadassah University Hospital,
Jerusalem, Israel. Scrapie strains ME7, 22A and 79A were kindly
provided by Dr. M. Groschup, Federal Research Centre for Virus
Diseases of Animals, Institute for Novel and Emerging Infectious
Diseases, Riems, Germany. Brains from 301V BSE mice were
obtained from Dr. M. Dawson, VLA Weybridge. Native scrapie
and normal sheep autopsy material was kindly provided by Dr. P.
Toumazos, Veterinary Services Laboratory, Cyprus.
For the tPA assay, 10% (w/v) homogenates were prepared using
a motor-driven homogenizer (Polytron Kinematica, Switzerland).
Two homogenization runs were performed (setting 4, 6 s each).
The homogenization buffer consisted of 0.5% w/v sodium
deoxycholate (DOC Fluka, Germany) and Igepal CA-630 0.5%
v/v (Sigma, St. Louis, MO) in PBS (phosphate-buffered saline, pH
7.4). For PrPCimmunoprecipitation and quantitative isolation of
PrPSc, 10% homogenates were prepared using a glass pestle and
mortar. The homogenization buffer was the same as above, the
only difference being the addition of Protease Inhibitors Cocktail
Mix at a final concentration of 0.1 v/v (Sigma, St. Louis, MO) in
the buffer used for the immunoprecipitation experiments.
Specific materials used for the tPA assay described below
include tPA and plasminogen, both of which were purchased from
Sigma, St. Louis, MO and the chromogenic substrate d-Ile-Pro-
Arg-p-nitroanilide dihydrochloride, which was acquired from
Chromogenix, Italy or Sigma, St. Louis, MO.
tPA assay
300 mg wet brain tissue (cerebella or brain stems for sheep and
cortices for mice and hamsters) was used to prepare homogenates.
Upon homogenization, the total protein content in each homoge-
nate was assayed, using the BCA protein assay kit (Pierce,
Rockford, IL). The homogenates were aliquoted and stored at
?70-C. The aliquots used for this study were not thawed more than
once.
Quantitative isolation of PrPScwas performed for every sample
(Polymenidou et al., 2002), and the presence of PrPScwas
confirmed by Western blotting.
The tPA activity assay was carried out in 96-well ELISA plates
(Greiner, Longwood, Fl). To each well were added: 30 Al distilled
water, 130 Al 0.1 M Tris–HCl, pH 8.0 (Sigma, St. Louis, MO),
0.1% v/v Tween 80 (Sigma, St. Louis, MO), 10 Al 8.4 AM
plasminogen, 10 Al 10% brain homogenate [diluted in 0.25% v/v
Triton X-100 (Sigma, St. Louis, MO) in order to contain 500 Ag
brain equivalent] and 20 Al 3 mM substrate d-Ile-Pro-Arg-p-
nitroanilide dihydrochloride. The assay was performed for each
sample in triplicates. Plates were incubated at room temperature for
3 h, and readings at 405 nm were performed using an ELISA plate
reader (MWG, Germany). Samples containing known amounts (0–
50 mIU) of tPA were also assayed. The OD405readings of those
samples were used to plot a standard curve and thus, to convert the
OD reads of the samples into specific tPA activity, expressed in
International Units (IU). Blank samples, containing water instead
of brain homogenates, were used as negative controls. The final
results were expressed as milli International Units of tPA per Ag of
total protein (mIU/Ag).
In order to optimize the assay, the kinetics of the reaction and
the effect of pH were investigated. For the kinetics study, the tPA
activity of a sample containing 500 Ag sheep brain equivalent was
measured every 5 min for 210 min and then after 24 h. As a
control, the tPA activity of the same sample was estimated in a
reaction mixture which contained no plasminogen. For the pH-
effect study, the pH of the reaction mixtures was adjusted with
NaOH or HCl to a variety of pH values, ranging from 6.0 to
approximately 12.0, and then the OD405was assessed after a 3
h incubation period at room temperature.
RNA isolation
Total RNAwas isolated from C57Bl mouse brains infected with
the 79A scrapie strain and normal C57Bl mouse brains, using a
commercially available kit (Promega, Madison, WI). All mice were
sacrificed at the same age of about 6 months, when the scrapie-
infected animals were in the final clinical stages of the disease. The
starting material for each RNA isolation was 50 mg cortex,
homogenized in 600 Al of denaturing buffer. The isolation was
performed following the manufacturer’s instructions. Isolated RNA
was resuspended in 60 Al RNase-free H2O and then treated with
RQ1 RNase-free DNase (Promega, Madison, WI) to remove co-
isolated DNA. Briefly, 6 Al of the RQ1 DNase and 7 Al of the
appropriate 10? reaction buffer were added to the RNA
suspension, and the mixture was incubated for 30 min at 37-C.
RQ1 DNase was inactivated by addition of 7 Al of stop solution
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containing 20 mM ethylene glycol-bis[h-aminoethyl ether]-
N,N,NV,NV,-tetraacetic acid (EGTA) pH 8.0 and incubation for 10
min at 65-C. To remove all compounds added for the DNase
treatment, RNA was purified on a Nucleospin RNA II column
(Macherey-Nagel, Germany), and it was finally eluted in 60 Al of
RNase-free H2O. The purified RNA was quantified and qualified
spectrophotometrically.
To assess the integrity of the isolated RNA, cDNA synthesis
was performed. PCR primers tPA1and tPA2were used (tPA1: 5V-
CCA CCT GTG GCC TGA GGC AGT ACA A-3V, tPA2: 5V-ATG
CCT CAT GCT TGC CGT AGC CAG A-3V). These primers
amplify a 473 bp fragment from the mouse tPA gene, when cDNA
serves as template. When genomic DNA is used as template, the
amplification product has a size of 750 bp because the genomic
sequence includes two introns in the genomic sequence (Siao et
al., 2003) not present in the cDNA. The absence of the genomic
DNA amplification product was ensured for all RNA preparations
by running the PCR products in a 1.5% tris boric acid EDTA
buffer, pH 8.0 (TBE) agarose gel and staining with ethidium
bromide.
Real-time PCR
cDNA was synthesized following a standard RT-PCR protocol,
using random hexamers as primers (Promega, Madison, WI) and
AMVreverse transcriptase (Promega, Madison, WI). Total RNA (4
Ag) was first denatured for 5 min at 70-C and then hybridized with
1 Ag of the random hexamers for 5 min on ice. cDNA was
synthesized for 1 h at 42-C by addition of the RT-PCR master mix
containing 5 U AMV, 50 mM Tris–HCl pH 8.3, 50 mM KCl, 10
mM MgCl2, 0.5 mM spermidine, 10 mM DTT and 1 mM of each
dNTP in a final reaction volume of 20 Al. The AMV reverse
transcriptase was inactivated by heating at 99-C for 5 min. For the
amplification of murine tPA and h-actin gene fragments, the
following primers were designed: mtPA117s: 5V-GGC AGA ACA
TAC AGG GTG GT-3V; mtPA117as: 5V-CTG CAG TAATGC GAT
GTC GT-3V; moACT139s: 5V-TGT TAC CAA CTG GGA CGA
CA-3V, moACT139as: 5V-CTG GGT CAT CTT TTC ACG GT-3V.
The tPA primers amplify a 117 bp DNA fragment, while the
primers for h-actin amplify a 139 bp DNA fragment. Real-time
PCR was carried out in the Opticon DNA Engine II (MJ-Research,
Reno, NV) as outlined: after initial denaturation for 3 min at 94-C,
the following cycle was repeated 40 times: 60 s at 94-C, 20 s at
62-C and 15 s at 72-C with a final extension of 3 min at 72-C.
SYBR Green I emission of the accumulating PCR product was
measured at the end of the extension step after each cycle. Melting
curve analysis was performed by measuring the emission of SYBR
Green I at 515 nm from 60-C to 90-C every 1-C after a 10 s hold at
each temperature. The PCR reaction mixtures contained cDNA
corresponding to 1.6 Ag of RNA, 2 U of the DyNAzyme
(Finnzymes, Finland), Mg2+-free buffer (10 mM Tris–HCl pH
9.0, 50 mM KCl 0.1% Triton X-100), 0.2 mM of each dNTP and
SYBR Green I (Sigma, St. Louis, MO) at a final concentration of
0.25?. PCR amplification products were visualized after separa-
tion on a 1.5% TBE agarose gel subsequently stained with
ethidium bromide.
PrPCimmunoprecipitation and tPA/plasminogen treatment
Immunoprecipitations were performed as previously described
(Sachsamanoglou et al., 2004). In brief, 10% brain homogenates
prepared as described in Tissue homogenates and specific reagents.
The equivalent of 10 mg brain tissue was centrifuged at 24,000 ? g
for 10 min. The supernatants were used for immunoprecipitations
with 4 Ag of the anti-PrP mAb 66.94b4, kindly provided by Dr. Jan
Langeveld, ID-Lelystad, The Netherlands and 25 Al of 50% Protein
G-Agarose beads (Upstate, Waltham, MA). The beads were
washed twice and finally resuspended in PBS prior to use. PrPC
treatment with 4.9 nM tPA and 0.28 AM plasminogen was
performed for 4 h at 37-C with agitation. The final volume of
the reaction was adjusted to 30 Al with PBS. The reactions were
stopped by adding SDS-PAGE sample buffer, and prion protein
was eluted from the beads by boiling. Proteins were separated on
an 18% SDS-PAGE gel and transferred onto a nitrocellulose
membrane (Gelman, East Hills, NY) for 2 h at 100 V. The
membrane was blocked with 5% w/v bovine serum albumin
(Sigma, St. Louis, MO) in PBST [PBS, 0.1% v/v Tween 20
(Sigma, St. Louis, MO)] and incubated with primary and secondary
antibody for 1 h each. The membrane was washed once for 15 min
and three times for 5 min with PBST between each antibody
incubation. The primary antibodies used to probe the membranes
were the anti-PrP monoclonal antibodies: 4F2 (0.44 mg/ml), which
recognizes an epitope on the N-terminal domain of PrP (51–90),
diluted 1:3500 in PBST; 12F10 (0.88 mg/ml), which recognizes an
epitope on the C-terminus domain of PrP (142–160), diluted
1:3500 in PBST; and 6H4 (1.00 mg/ml, Prionics, Switzerland),
which recognizes an epitope located at the C-terminal PrP domain
(144–152), diluted 1:5000 in PBST. Both 4F2 and 12F10
antibodies were kindly provided by Prof. Walter Bodemer,
Goettingen, Germany (Krasemann et al., 1996). The membranes
were incubated with rabbit anti-mouse alkaline phosphatase
conjugated secondary antibody (Pierce, Rockford, IL) and devel-
oped with the CDP star reagent (NEB, Beverly, MA) following the
manufacturer’s instructions.
PrPScquantitative isolation and tPA/plasminogen treatment
10% w/v brain homogenates, prepared as previously described,
were centrifuged at 24,000 ? g for 10 min. The supernatants were
used for PrPScisolation, as described elsewhere (Polymenidou et
al., 2002). After the addition of PMSF, the samples were left
overnight at 4-C. The resulting pellets were resuspended in 20 Al of
PBS and treated with 4.9 nM tPA and 0.28 AM plasminogen for 4
h at 37-C with agitation. The reactions were stopped by adding
SDS-PAGE sample buffer, and the samples were boiled for 10 min
before loading on an 18% SDS-PAGE gel. Electro-transfer and
Western blotting of the proteins with either anti-PrP mAb 4F2,
diluted 1:3500 in PBST, or anti-PrP mAb 6H4, diluted 1:5000 in
PBST, were performed as described above.
In vivo experiments
The wild-type C57Bl6 mice used were purchased originally
from Jackson Laboratories and bred at SUNY Stony Brook. The
tPA-deficient (tPA?/?) animals (Carmeliet et al., 1994) were
generated on a 129 background and have subsequently been
backcrossed to the C57Bl6 background for 12 generations. The
plasminogen-deficient (plg?/?) mice (Bugge et al., 1995) original-
ly purchased from Jackson Labs, and bred in SUNY Stony Brook
are also in the C57Bl6 background.
Groups of 4 adult mice of either sex, C57Bl wild type, tPA?/?
or plg?/?, weighing approximately 25 g, were injected intraper-
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itoneally with atropine (0.6 mg/kg of body weight) and then were
deeply anesthetized with 2.5% avertin (0.02 ml/g of body weight).
The mice were placed in a stereotaxic apparatus. An incision was
made over the sagittal suture, and the skin was retracted with
micro-clips. A small burr hole was drilled at the stereotaxic
coordinates of the dorsal hippocampus (bregma ?2.5 mm,
medial–lateral 1.7 mm and dorsoventral 1.6 mm). The mice were
inoculated with 1 Al of 1% w/v brain homogenate in 0.9% saline.
These homogenates were ME7 and 22A scrapie-infected brain
homogenates and the respective normal non-infected homogenate
from age-matched mice. Mice were then monitored for the
progression of disease, evaluating the time of appearance of
clinical symptoms, as well as the time of death.
After variable lengths of time (before and as the clinical
symptoms appear, usually between 8–23 weeks), the animals
were anesthetized and perfused through the heart with PBS
followed by 4% paraformaldehyde. The brains were removed,
post-fixed in PBS containing 4% paraformaldehyde and 30%
(w/v) sucrose for 24 h and then processed for embedding in
paraffin. The paraffin sections were immersed in concentrated
formic acid, washed and then autoclaved. The presence of PrPSc
deposition was visualized by immunohistochemistry using the
rabbit polyclonal antibody 78295 (a gift from Dr. R. Kascsak,
Institute for Basic Research in Developmental Disabilities, Staten
Island, NY) using a previously described protocol (Manousis et
al., 2000; van Keulen et al., 1999). Furthermore, using hematox-
ylin counterstaining, we evaluated the tissue for the presence of
extensive vacuoles.
Results
tPA assay
There is mounting evidence for an interaction between prion
protein, plasminogen and tPA (Fischer et al., 2000; Maissen et
al., 2001; Praus et al., 2003; Ryou et al., 2003), while urokinase
plasminogen activator does not seem to associate with PrP (Ellis
et al., 2002). Prompted by these observations, we investigated
the tPA activity in TSE-infected brain tissue. We studied the
tPA activity in infected brain homogenates, relative to appro-
priate control homogenates using an indirect chromogenic assay
based on the proteolytic activity of plasmin. Plasmin is
produced by the hydrolysis of the plasminogen and it cleaves
a peptidic bond (Arg-p-nitroanilide) of the substrate D-Ile-Pro-
Arg-p-nitroanilide producing p-nitroaniline. p-Nitroaniline is
soluble and exhibits a strong absorption at 405 nm that is
linear with increasing tPA concentrations over a broad working
range.
Our preliminary work indicated that at least 500 Ag brain
equivalents are required for linear kinetics. The time course of
tPA activity is sigmoid with a lag period of 40–45 min. The
reaction is linear for the time period from 45 up to 180 min and
reaches a plateau after approximately 3 h. All subsequent mea-
surements were made after 180 min. Interestingly enough, in
control (blank) reactions, containing plasminogen but no tPA
enzyme source, the OD increases linearly with time, even after 3
h, indicating that the substrate is degraded. On the other hand,
blank samples, which contained no exogenous plasminogen, did
not exhibit enzymatic activity over the time course that the
measurements were taken (data not shown). Since the substrate
cannot be hydrolyzed by tPA or other proteolytic enzymes which
may be present in the homogenate and it is stable throughout the
time period the readings were performed, the observed OD
increase in reactions containing only plasminogen should be
attributed to spontaneous hydrolysis of plasminogen to plasmin.
Hence, as the addition of plasminogen is mandatory in order to
achieve the desired results, the OD readings must be performed
within 2 and 3 h in order to rule out the effect of the spontaneous
hydrolysis of plasminogen.
tPA and plasmin are serine proteases, and as such, their
activity is pH-dependent. After a detailed investigation, we
concluded that the tPA-mediated plasmin reaction is pH-sensitive
and that, although significant enzymatic activity is obtained at
various pH settings, ranging from 7.5 to 9.0 without significant
effects on tPA activity, the optimal pH value setting is 8.0 (data
not shown).
To assess the possibility of inactivation of tPA during the
process of sample preparation, two homogenization methods were
evaluated, the one using a motor-driven homogenizer and the other
using a glass pestle and mortar. The homogenization buffer in both
procedures was the same. When the OD values were normalized
per microgram of protein, both protocols revealed similar tPA
activity, and the specific activity of tPA was not affected,
independent of the employed method. Clearly, both methods are
reliable and efficient and both of them can be used, depending on
the experimental needs and tissue availability. Homogenization
with the pestle and mortar is more versatile and suitable for smaller
amounts of tissue.
Significant elevation of tPA activity in different species,
including hamsters (experimentally infected with scrapie), mice
(experimentally infected with scrapie and BSE) and naturally
occurring scrapie sheep, was observed, compared to the respective
normal animals (Fig. 1). Preliminary data also imply elevated tPA
activity in mice experimentally infected with BSE (301V BSE
mice, data not shown).
Variation of tPA activity in sheep scrapie clinical cases can be
attributed to different stages of the disease, age, brain region, tissue
processing or handling. An interesting observation was that
hamster tissue has significantly lower tPA activity than the other
species.
Real-time PCR
In parallel experiments, tPA-gene expression was monitored by
real-time PCR. Beta-actin was used as a reference gene.
Amplification of the expected fragments as single products was
ensured by melting curve analysis (data not shown) and agarose gel
electrophoresis (Figs. 2A, B). In all samples, the actin fragment
amplification was observed first, while tPA cDNA amplification
began about 7–8 cycles later. The relative expression of the tPA
gene, observed to be approximately 10% higher in the infected
animals compared to the normal ones (Fig. 2C), is in good
agreement with the obtained results on tPA activity. Relative
expression of the tPA gene in both samples was estimated using the
REST software (Pfaffl et al., 2002).
Incubation of PrPCand PrPScwith exogenous tPA and
plasminogen
To investigate whether elevated tPA activity is related to PrP
metabolism, we investigated if tPA-generated plasmin could cleave
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prion protein in vitro. Immunoprecipitated prion protein originat-
ing from normal sheep brain homogenates was precipitated with
anti-PrP mAb 66.94b4 (manuscript in preparation). The isolated
PrPCwas incubated with exogenous tPA and plasminogen at
concentrations of 4.9 nM and 0.28 AM respectively. Previous
reports demonstrated that, when recombinant prion protein was
treated with tPA and plasminogen, PrP was cleaved at residue 110,
resulting in an N-terminal (aa 23–110) and a C-terminal fragment
(aa 111–231). The N-terminal fragment was shown to accelerate
plasminogen activation, suggesting that this mechanism could
represent a regulatory mechanism of pericellular proteolysis
(Kornblatt et al., 2003; Praus et al., 2003). Following the
experimental protocol, immunoprecipitated PrPCfrom normal
sheep brain homogenates was digested with exogenous tPA and
plasminogen. Using antibodies that recognize different epitopes of
prion protein, we detected an N-terminal fragment with anti-PrP
mAb 4F2 (Fig. 3, lanes 1 and 2; arrow indicates the expected
fragment) and three C-terminal fragments, resulting from the
digestion of double-, mono- and non-glycosylated PrPC(Fig. 3,
lanes 3 to 6). The expected molecular weights, indicated by arrows,
are 26 kDa, 22 kDa and 16.5 kDa respectively. These fragments do
not result from the cleavage of anti-PrP mAb 66.94b4 that was
used for PrPCimmunoprecipitation, as the antibody remains
undigested after treatment with tPA and plasminogen (data not
shown).
PrPScenriched preparations were similarly treated with exo-
genous tPA and plasminogen to determine if the pathological
isoform was similarly digestable. PrPScquantitative isolation
was performed from scrapie-infected sheep brain homogenates
in the presence or absence of proteinase K. The epitope recog-
nized by anti-PrP mAb 4F2 is destroyed during treatment with
proteinase K, and this antibody should not detect the remaining
proteolytic-resistant fragments of prion protein (Fig. 4, lanes 3
and 4), while the epitope recognized by anti-PrP mAb 6H4 is
unaffected by proteinase K treatment. The isolated PrPScwas
not cleaved in the presence of tPA and plasminogen, as no
truncated forms of PrPScwere observed with either of the anti-
bodies (4F2 and 6H4) used for the detection of smaller frag-
ments (Fig. 4).
In vivo experiments: inoculation of wild-type, tPA-deficient and
plasminogen-deficient mice with PrPScbrain homogenates
Since it was reported that the misfolded PrPSc, but not the
normal cellular protein PrPC, could bind to plasminogen (Fischer et
al., 2000), we decided to assess in vivo whether plasminogen could
actively participate in pathogenesis, hypothesizing that it might aid
PrPCto misfold into PrPSc. If that were the case, we would expect
that mice deficient in plasminogen would also be deficient in the
conversion of PrPCto PrPScand would not develop disease or
show a significant delay in the progression of the disease and the
appearance of clinical symptoms.
Fig. 2. Real-time PCR data. (A) RT-PCR amplification products of the tPA
gene using the primers tPA1and tPA2. Lane 1: template RNAwas not RQ1
RNase-free DNase treated. Lane 2: RT-PCR amplification product of RQ1
RNase-free DNase treated total RNA, as described in the Materials and
methods section. (B) Real-time PCR amplification products representative
for all reactions. Lane 1: h-actin fragment. Lane 2: tPA fragment. (C)
Relative expression of the tPA gene resulted from real-time PCR data
analysis, normalized data using h-actin as reference gene. tPA gene
expression in normal animals (control) compared to 79A mouse scrapie-
infected animals at the terminal stage of the disease. Mean values of three
normal and three scrapie samples, each one in triplicate. Bars represent the
standard deviation of the samples. Relative expression was estimated by
using the REST software (Pfaffl et al., 2002).
Fig. 1. Comparison of tPA activity in TSE and uninfected brain tissue from
different species. tPA activity, expressed as mIU of tPA per microgram of
total protein, was measured as described in Materials and methods. The
sheep samples are from the brain stem and cerebellum of animals naturally
infected with scrapie or control sheep. tPA activity of mice and hamster
samples was measured from whole brain homogenates of animals
experimentally infected with TSEs and control animals. The data are
analyzed by a mixed effects model with the healthy-scrapie factor a fixed
effect and the animal factor, which is nested within the healthy-scrapie
factor, a random effect. The ANOVA statistics indicates that there is a
significant difference (P < 0.05) between the scrapie and the healthy
animals for the hamster and the sheep brain stem samples. P values in mice
and sheep cerebellum are 0.13 and 0.16 respectively indicating similar
pattern.
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Three different strains of mice were challenged: C57Bl wild-
type, tPA-deficient and plasminogen-deficient. Four mice from
each genotype were injected with ME7 and 22A brain homo-
genates (Figs. 5A and B) respectively. Mice were then monitored
for the progression of disease, evaluating the time of appearance
of clinical symptoms, as well as the time of death. The clinical
symptoms observed included muscle strength weakness resulting
in ataxia, hunched posture, ocular secretion and eventually death.
Routine immunohistochemistry was performed for the detection of
PrPScdeposition and vacuolization (data not shown). As it is
demonstrated in Fig. 5, the plasminogen-deficient mice developed
symptoms of disease and succumbed to it faster than the tPA-
deficient or wild-type animals, irrespective of the strain of the
infectious scrapie material. All the strains of mice injected with
ME7 showed a faster progression of disease in general, but well
within the timing reported in the literature (Walsh et al., 2000).
Mice of all three genotypes injected with normal (uninfected)
brain homogenate did not show any clinical symptoms, thus ruling
out technical or non-specific problems with the transgenic
animals.
Discussion
tPA is a multifunctional molecule, directly involved in normal
functioning of the nervous system (Strickland, 2001) but also
related to neuronal cell death (Tsirka, 1997; Tsirka et al., 1996).
Relation of tPA to prion diseases (Ellis et al., 2002; Fischer et al.,
2000; Kornblatt et al., 2003; Maissen et al., 2001; Praus et al.,
2003) and other neurodegenerative diseases (Ledesma et al., 2000;
Lu et al., 2002) has previously been reported. Little notice, though,
has been given to the regulation of tPA activity during the course of
prion diseases. In our study, an upregulation of tPA levels is
demonstrated in parallel with the occurrence of prion diseases.
Furthermore, it was found that transgenic mice, lacking the tPA or
the plasminogen gene, are more susceptible to the TSEs than their
wild-type counterparts. Our data therefore imply a neuroprotective
rather than a neurotoxic role for tPA.
Although age and gender do not seem to affect tPA activity
significantly (Eliasson et al., 1993), a serious effort was made to
use age- and sex-matched animals in the tPA activity and gene
expression studies. The observed increase in tPA activity is
Fig. 4. PrPSctreatment with exogenous tPA and plasminogen. PrPScfrom sheep scrapie samples was enriched and examined for resistance to plasmin-mediated
proteolysis as described in the Materials and methods section. Lanes 1, 2, 5, 6: Proteinase K untreated samples; lanes 3, 4, 7, 8: proteinase K treated samples.
Each group of samples was incubated with or without 4.9 nM tPA and 0.28 AM plasminogen for 4 h at 37-C and then analyzed by immunoblotting. Lanes 1, 2,
3 and 4 were probed with anti-PrP mAb 4F2, while lanes 5, 6, 7 and 8 were probed with anti-PrP mAb 6H4. Dots indicate double-, mono- and non-glycosylated
PrP, while arrows indicate double-, mono- and non-glycosylated proteinase K resistant PrP.
Fig. 3. PrPCtreatment with exogenous tPA and plasminogen. PrPCwas immunoprecipitated from sheep homogenates with the anti-PrP mAb 66.94b4. Lanes 1,
3, 5: control immunoprecipitated PrPC; lanes 2, 4, 6: immunoprecipitated PrPCtreated with 4.9 nM tPA and 0.28 AM plasminogen for 4 h at 37-C and then
analyzed by immunoblotting. Blots were immunostained with: anti-PrP mAb 4F2 that recognizes residues 51–90 of PrP molecule (lanes 1, 2), anti-PrP mAb
6H4 that recognizes residues 144–152 (lanes 3, 4) and anti-PrP mAb 12F10 which binds to residues 142–160 (lanes 5, 6). Dots indicate undigested double-,
mono- and non-glycosylated immunoprecipitated PrPC, while arrows show the PrP fragments after treatment with tPA and plasminogen.
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consistent with the real-time PCR results, where tPA mRNA in
79A scrapie terminally ill mice is found to be expressed at a level
about 10% higher than in normal mice. The upregulation of such a
molecule could be used as an additional biochemical marker in
coordination with more studies to further analyze its role and its
involvement in TSE pathogenesis and other neurodegenerative
diseases.
Furthermore, we tried to identify the function of elevated tPA
activity in prion diseases by studying the impact of tPA-generated
plasmin on prion protein. Several studies for the role of the
plasminogen system on the metabolism of prion protein have been
published (Ellis et al., 2002; Kornblatt et al., 2003; Praus et al.,
2003), but all were based on use of recombinant PrP. In our study,
immunoprecipitated PrPCfrom normal sheep brain homogenates
was used, as well as PrPScquantitatively isolated from scrapie-
infected sheep brain homogenates. Our experiments show cleavage
of PrPC, in accordance with the reported digestion of recombinant
PrP (Kornblatt et al., 2003; Praus et al., 2003), whereas cleavage of
PrPScunder similar reaction conditions failed or at least was not
detectable under our experimental conditions.
It is already known that PrPCstimulates tPA-mediated plasmin
generation (Praus et al., 2003). A possible explanation for this
phenomenon could be the binding of both proteases on PrP via
their kringle domains (Ryou et al., 2003). It is further known that
PrPScbinds tightly to plasminogen (Fischer et al., 2000; Maissen et
al., 2001). The reported failure of tPA-generated plasmin to cleave
PrPSccould be explained by possible inaccessibility of plasmin due
to the supramolecular conformational changes of PrPSc.
Given the in vitro interaction between the misfolded prion
protein and plasminogen, a unique handle was offered to look at
the molecular mechanisms that follow prion misfolding by using
mice deficient in plasminogen or tissue plasminogen activator. To
address the question if and how the tPA/plasminogen system is
implicated in prion diseases, we performed a bioassay by
inoculating tPA- or plasminogen-deficient mice with two different
mice scrapie strains. Our in vivo results with tPA- and plasmin-
ogen-deficient animals demonstrated a direct impact on the course
of the disease, suggesting that a potential assignment to plasmin-
ogen, as implied by Fischer et al. (2000), would not be appropriate
since plasminogen-deficient animals did develop symptoms and
eventually died. To the contrary, incubation period in plasminogen-
deficient mice was significantly shorter than their tPA-deficient or
wild-type counterparts. The elevated tPA activity and tPA gene
expression in combination with our in vivo results may argue in
favor of a neuroprotective action of tPA, similar to the one
described for Alzheimer disease, where the elevated tPA activity
seems to correlate with the plasmin cleavage of beta-aggregates
(Ledesma et al., 2000). Our results from the in vitro incubation of
PrPScwith tPA/plasminogen, however, rule out the possibility that
tPA or plasminogen (via their kringle domains) may act to bind and
remove misfolded PrPScfrom its sites of accumulation during early
stages of disease.
The elevated tPA activity observed in our study may be linked
to neuroprotection against TSEs through proteolytic processing of
the PrPC, which was found in vitro to be sensitive to the effects of
the tPA/plasminogen system. The primary structure of the prion
protein is well conserved over many mammalian species (van
Rheede et al., 2003), and the 106–126 PrP fragment has been
shown to be necessary for the conversion of PrPCto PrPSc
(Tagliavini et al., 1993). Since PrP cleaved at position 110 is a less
suitable substrate for PrPScreplication, tPA/plasminogen-mediated
cleavage of PrPCat position 110 could act protectively against the
conversion of PrPCto the pathological isoform [also discussed in
Praus et al. (2003)].
Another hypothesis is that tPA may provide nonproteolytic
neuroprotection. It has been shown that tPA attenuates oxidative-
stress-related neuronal death and that this action is independent of
plasminogen activation (Kim et al., 1999). Taking into consider-
ation that oxidative stress is a key event in the neuropathology of
prion diseases (Budka, 2003; Guentchev et al., 2002; Hur et al.,
2002), tPA overexpression in tissues infected with TSEs might
represent a response to this stress factor. However, since
plasminogen-deficient mice seem to be more susceptible to TSEs
than their tPA-deficient counterparts, tPA’s nonproteolytic neuro-
protection mechanisms should be considered to be of lesser
importance.
Up to this point, our results, including elevated tPA activity
during TSE pathogenesis and significant reduction in TSE
incubation time in tPA-deficient mice, indicate that tPA has a
protective rather than a neurodegenerative role. Of course, we
cannot rule out the possibility that a more complex phenom-
enon is taking place including other molecules with a simul-
taneous protective and destructive role of the tPA/plasminogen
system.
We strongly believe that more detailed studies will contribute
significantly to a better understanding of the precise relationship
between the tPA/plasminogen system and TSE pathogenesis,
yielding further knowledge of these pathogens that will lead in
the development of novel therapeutic approaches.
Fig. 5. Plasminogen-deficient mice are more susceptible to intracerebral
injection of prion infectious material than tPA-deficient or wild-type mice.
Four animals from each of three different mouse strains (C57Bl, tPA-
deficient or plasminogen-deficient) were inoculated with 1 Al of 1% (w/v)
brain homogenate from mice infected with the ME7 (A) or 22A (B)
experimental scrapie strains or with homogenates prepared from age- and
strain-matched non-infected mice. None of the mice inoculated with control
homogenates showed disease symptoms over the course of the experiment.
The columns show the average time from scrapie inoculation to death, with
the bars representing the standard deviation.
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Acknowledgments
The authors wish to thank Dr. C.H. Panagiotidis (CERTH-INA)
for the revision of the manuscript, assistant professor A. Nicolaou
(University of Macedonia) for the statistical analysis of the data
and K. Pasentsis (CERTH-INA) for helpful suggestions on the RT-
PCR work. We also would like to thank the members of Dr. Tsirka-
lab for their helpful suggestions. This study was partially supported
by the European Union through QLK2-CT-2002-81523 TSE LAB,
QLK2-CT-2001-01924 and the Greek Ministry of Health through
the Center for Control of Infectious Diseases KEEL. S.E. Tsirka
was partially supported by an NIH/NINDS grant.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.nbd.2005.04.008.
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