Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 6097, Centre National de la Recherche Scientifique. 660, route des lucioles, 06560 Valbonne, France
Author for correspondence (e-mail:
chabry{at}ipmc.cnrs.fr)
Accepted 18 March 2003
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Summary |
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Key words: Transmissible spongiform encephalopathy, Prion, siRNA, Scrapie, PrP-res, PrPsc, PrP-sen, Prnp
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Introduction |
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Chronically scrapie-infected cell lines have been extensively used as
models for selecting `anti-prion' drugs such as porphyrines
(Caughey et al., 1998),
amphotericin B (Mange et al.,
2000
) and quinacrine (Doh-Ura
et al., 2000
). Since PrP-sen expression is essential for both TSE
pathogenesis and conversion leading to PrP-res accumulation, we studied the
effect of specific Prnp gene silencing molecules in scrapie-infected
neuroblastoma cells (N2aS12sc+). Small interfering RNAs (siRNAs)
provide new powerful tools to potentially silence any targeted gene
(Elbashir et al., 2001
).
Because siRNA duplexes trigger specific gene silencing in mammalian somatic
cells without the activation of any unspecific response, the analysis of gene
function in cultured cells has now become possible. Here, we show that
transient transfection of siRNA duplexes designed from the mouse Prnp
gene (moPrnp) causes a rapid loss of PrP-sen expression and abrogates
PrP-res formation in N2aS12sc+ cells. By contrast, the scrambled
siRNA failed to inhibit both the PrP-sen expression and PrP-res accumulation
in these cells suggesting a highly specific siRNA effect. Finally, we
demonstrate that the siRNA gene silencing is independent of both the host cell
types and mouse-adapted scrapie strain.
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Materials and Methods |
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Cell cultures
Neuroblastoma N2a cells over-expressing mouse PrP (subclone #58) were
cultured in Opti-MEM containing 10% inactivated FBS (FBSi),
penicillin-streptomycin and 1 mg/l G418. This cell line named
N2aS12sc+ has been chronically infected with brain homogenates of
Chandler strain-infected mice (Nishida et
al., 2000). Since the treatment of N2aS12sc+ cells with
Congo Red (1 µg/ml) totally cured the cells of PrP-res, Congo Red-treated
N2aS12 cells were used as uninfected cells (N2aS12). The GT1 cells, infected
with brain homogenates of 22L murine scrapie strain, were grown in Dulbecco
modified Eagle's medium containing 5% fetal calf serum, 5% horse serum, and
penicillin-streptomycin. All cultured cells were maintained at 37°C in 5%
CO2 and split 1:4 every 4 days.
siRNA preparation and transfection
siRNAs corresponding to the moPrnp gene from codon 392 to 410 were
synthesized by Eurogentec (Seraing, Belgium). Typically, siRNAs were made of
19 ribonucleotides followed by two extra thymidine bases at the 3' end
overhang on both strands. The specific siRNAs sequences used were:
5'-GCC-CAG-CAA-ACC-AAA-AAC-CTT-3' (sense) and scramble
5'-CGC-ACC-AGA-ACA-AAC-ACA-CTT-3' (sense). The specific siRNA
sense 5'-GCC-CAG-CAA-ACC-AAA-AAC-CTT-3' was labeled with
6-carboxyfluorescein (6-FAM) on the 3' end. Annealing for duplexes siRNA
formation was performed by incubation at 92°C for 1 minute in 50 mM
Tris-HCl pH 7.5 containing 100 mM NaCl, followed by a 60 minute incubation at
room temperature. The final stock concentration of siRNA duplexes (20 µM)
was stored at 4°C until use.
Twenty-four hours before the transfection, cells were seeded at 10-15% confluence in a 12-well culture plate with appropriate culture medium. Varying amounts of double strand siRNA (1-10 µl) were mixed with the corresponding half-volume (0.5-5 µl) of OligofectamineTM reagent for 20 minutes according to the manufacturer's instructions. The mixture was then applied to the cells in a final volume made up to 500 µl with Opti-MEM without FBSi and antibiotic. After incubation for 4 hours at 37°C under 5% CO2, 250 µl of Opti-MEM supplemented with 30% FBSi and a penicillin/streptomycin mixture were added. Cells were then cultured for three days at 37°C until confluent.
Assay for PrP-res accumulation in N2aS12sc+
Confluent cultures were lysed for 10 minutes at 4°C in lysis buffer (50
mM Tris-HCl pH 7.4 containing 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium
deoxycholate, 5 mM EDTA), then centrifuged for 5 minutes at 3000
g. For detection of PrP-sen, one-tenth of post-nuclear
supernatants were directly mixed with the same volume of 2x denaturing
loading buffer. For detection of PrP-res, samples were digested with 20 µg
of PK per mg of total proteins for 30 minutes at 37°C. The digestion was
stopped by adding Pefabloc (1 mM) for 5 minutes before centrifugation at
20,000 g for 90 minutes at room temperature. Pellets were
resuspended in 30 µl of denaturing loading buffer, sonicated, boiled for 5
minutes and loaded onto a 12% polyacrylamide gel. Proteins were separated by
SDS-PAGE, then electroblotted onto a nitrocellulose membrane (Protran BA83,
Schleicher & Schuell). Membranes were treated with a solution containing
5% nonfat dried milk in 20 mM Tris-HCl pH 8, 100 mM NaCl, 0.1% Tween 20, and
incubated overnight at 4°C with the appropriate primary antibody. Blots
were developed by using an enhanced chemiluminescence system (Pierce,
Rockford, IL) and exposed on X-ray films (X-OMAT AR, Kodak). Densitometry was
performed with `National Institutes of Health' IMAGE software, on
at least four independent experiments and results expressed as a percentage of
control levels.
Confocal microscopy
N2aS12 cells were grown on glass coverslips (Lab-Tek) in Opti-MEM
supplemented with 10% FBSi. Cells were transfected as described above with the
6-FAM-conjugated siRNAs duplexes (400 nM) or the single-stranded
6-FAM-conjugated siRNA as a control experiment. At the indicated
post-transfection times, coverslips were washed twice with phosphate-buffered
saline (PBS) and fixed in paraformaldehyde 3% in PBS for 10 minutes at room
temperature. Cells were washed twice with PBS and then incubated in PBS
containing 50 mM NH4Cl for 10 minutes in order to quench excess
free aldehyde groups. Cells were permeabilized in PBS containing 0.1% Triton
X-100 and 1 µg/ml propidium iodide for 10 minutes at room temperature.
Coverslips were washed twice with PBS, and then mounted on glass slides with
mowiol. Cells were analysed using a laser scanning confocal inverted
microscope (Leica, TCS-SP) equipped with an argon-krypton laser. Samples were
scanned under both 488 nm and 568 nm excitation wavelength for
6-FAM-conjugated siRNA and for propidium iodide, respectively. Images were
acquired as single transcellular optical sections and averaged over at least 8
scans per frame.
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Results |
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To assess whether the nature of the host cell or the scrapie strain could
influence the siRNA inhibition effect, siRNAs were transfected into the mouse
hypothalamic cell line (GT-1) chronically infected with the mouse scrapie
strain 22L (Nishida et al.,
2000). The detectable amounts of PrP-res were also drastically
reduced in this cell line after transfection with Prnp
gene-specific siRNA duplexes (Fig.
2) and IC50 values were comparable with those obtained
using N2aS12sc+ cells (IC50
106 nM). This suggests
that neither host cell types nor mouse scrapie strains influence the
gene-silencing effect of the siRNA. During the time course of these
experiments (4 days), no cytotoxic effect of siRNAs was observed.
|
To estimate the efficiency of siRNA transfections into the neuroblastoma
cells, confocal microscopy analysis was performed using fluorescein-labeled
siRNA duplexes (Fig. 3). The
percentage of fluorescein-labeled neuroblastoma cells (green) reached
82%±6% as early as 48 hours post-transfection and was stable over
72 hours (Fig. 3A). No green
fluorescence was detected in cells transfected with the fluorescein-labeled
single-stranded RNA as shown in the control experiment
(Fig. 3A). We also used
confocal imaging to investigate the sub-cellular localization of the
fluorescein-labeled siRNA duplexes. Transfected cells exhibited a punctate
fluorescence pattern largely localised to the perinuclear cytoplasmic region
(Fig. 3B). No labeling was
observed at the cell surface or inside the nucleus. Based on both the
transfection efficiency and the assessment of PrP expression, we found that
OligofectamineTM was the most efficient transfection reagent for siRNA
duplexes.
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To determine whether the PrP-res depletion induced by siRNA was transient or permanent, chronically infected N2aS12sc+ cells were transfected once with 400 nM siRNA and then cultured for either one and two weeks through two and four passages, respectively. Samples were assayed by Western blot analysis for the presence of PrP isoforms in the presence or in the absence of PK-digestion (Fig. 4). PrP-sen was barely detectable in transfected cells before passaging but levels increased with the number of passages, reaching the level observed with untreated-cells after four passages. A single siRNA transfection led to a complete disappearance of PrP-res immunoreactivity after two passages of N2aS12sc+ cells. However, after four passages, low levels of the PK-resistant PrP isoform were observed in N2aS12sc+ cells. Our data suggest that a single exposure of N2aS12sc+ cells to siRNA was not sufficient to completely remove the pathogenic isoform and permanently suppress scrapie infection.
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Discussion |
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The factors responsible for the rate and the strength of siRNA-induced gene
suppression are currently unknown. Here, we demonstrate that siRNA designed
from the moPrnp gene was highly specific to cells expressing the PrP
mouse sequence. The siRNA effect was independent of both the scrapie strains
and the host cell type. Furthermore, no difference in siRNA efficiency was
observed between moPrP overexpressing cells (N2aS12 subclone) and N2a cells
expressing only endogenous PrP (data not shown). The finding that exposure of
chronically infected neuroblastoma cells to siRNA results in undetectable
amounts of PrP-res after 3-4 days, suggests that these cells express proteases
that degrade PrP-res aggregates. Recently, Enari et al. have proposed that the
cellular level of PrP-res is determined by the rate of its formation from the
substrate, PrP-sen, and its catabolism
(Enari et al., 2001). The
removal of PrP-sen could displace the equilibrium to the degradation
processes. An obvious limitation is that although PrP-res levels were
dramatically decreased by acute siRNA exposure, the cells were not
definitively `cured' of scrapie infection; thus, chronic treatments or longer
exposures of siRNA may be considered in the future. We are currently
performing experiments to improve the long-term effects of siRNA duplexes.
Interestingly, a recent report has shown the selective suppression of gene
expression induced by siRNA in primary mammalian neurons
(Krichevsky and Kosik, 2002).
The role of PrP and its interaction with putative partners in neurons have not
yet been clarified. Thus, siRNA technology should provide insights into the
understanding of the physiological functions of PrP in somatic cultured
cells.
Although there is likely to be technical difficulties with siRNA delivery
in vivo, its use to inhibit gene expression in mice has recently been reported
(Lewis et al., 2002). Although
efficient and powerful brain delivery systems remain to be found, it appears
likely that siRNA technology could constitute a new promising strategy for
therapy against TSEs. Current drug treatments delay, but do not prevent, the
appearance of clinical symptoms and death in experimental scrapie-infected
animals (Demaimay et al., 1997
;
Priola et al., 2000
). The in
vivo use of siRNA alone, or in combination with these drugs, should be
considered to improve the life span and cure sick animals. The recent
development of siRNA technology offers a powerful tool to ablate specific gene
expression in a wide range of mammalian cells including neurons.
Our results show that the siRNA technique should be useful for dissecting basic mechanisms of prion pathogenesis, in addition to its obvious potential as a therapeutic agent. Ultimately it may give effective results in therapy of numerous neurodegenerative diseases linked to expression of misfolded endogenous proteins such as Alzheimer's, Huntington's and Parkinson's diseases.
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Acknowledgments |
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Footnotes |
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