Stec1 1 Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland;2Gene Function Research Laboratory, National Institut
Trang 1Efficient inhibition of b-secretase gene expression in HEK293 cells
ribozymes
Barbara Nawrot1, Slawomir Antoszczyk1, Maria Maszewska1, Tomoko Kuwabara2, Masaki Warashina2, Kazunari Taira2and Wojciech J Stec1
1
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland;2Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba Science City, Japan
The b-amyloid peptide (Ab) is a major component of
toxic amyloid plaques found in the brains of patients with
Alzheimer’s disease.Ab is liberated by sequential cleavage
of amyloid precursor protein (APP) by b- and
c-secre-tases.The level of Ab depends directly on the hydrolytic
activity of b-secretase.Therefore, b-secretase is an
excel-lent target for drug design.An approach based on
RNA-cleaving ribozymes was developed to control expression
of b-secretase.Two sites of mRNA coding b-site APP
cleaving enzyme were chosen as target sequences for
endogenously delivered ribozymes.The ribozyme cassette
was designed to constitute a catalytic hammerhead core
and substrate recognition arms, flanked at the 5¢-terminus
by tRNAVal and at the 3¢-terminus by constitutive
transport element sequences.Ribozyme cassettes were cloned into a pUC19 plasmid and used for transient transfection of HEK293 cells.We demonstrate that such ribozymes efficiently inhibit b-secretase gene expression at both the mRNA (up to 95%) and the protein (up to 90%) levels.Inhibition of b-site APP cleaving enzyme activity directly influences the intra- and extracellular population of Ab peptide.Therefore, such ribozymes may be considered as molecular tools for silencing the b-secretase activity, and further, as therapeutic agents for anti-amyloid treatment
Keywords: Alzheimer’s disease; hammerhead ribozyme; b-secretase
Alzheimer’s disease (AD) is a neurodegenerative disorder
characterized by progressive deposition of senile plaques in
the brain.The major component of these aggregates is a
4-kDa b-amyloid peptide (Ab), a product of proteolytic
cleavage of the amyloid precursor protein (APP) [1].The
processing of the transmembrane precursor glycoprotein
APP in vivo occurs by two different pathways.A
conven-tional nonamyloidogenic pathway occurs via proteolytic cleavage of APP by a- and c-secretases and results in release
of nontoxic soluble a-APP(s) protein and two other shorter products P3 and C7 [2,3].In normal healthy individuals these products protect neuronal cells against oxidative stress and participate in wound repair [4–7].Another kind of APP processing prevails in the brains of AD patients with ageing-related dementia.In such cases the cleavage of APP occurs
on the amyloidogenic processing pathway.APP is cleaved at the N-terminus of the Ab region by b-secretase and at the C-terminus by c-secretase.The product of these cleavages is
a 39–43-amino acid b-amyloid peptide.The major cleavage products are Ab40 and Ab42.According to the amyloid hypothesis, accumulation of Ab in the brain is the primary influence driving AD pathogenesis [8].The gene encoding b-secretase was sequenced recently [9–13].It is an aspartyl protease 2 (Asp2), also called b-site APP cleaving enzyme (BACE) or memapsin 2.Several approaches have been undertaken to find an effective inhibitor for human b-secretase activity [9,14–17].The selectivity of peptidomi-metic inhibitors, however, is limited due to their affinity toward other cellular aspartyl proteases [14].It has been shown that BACE knockout mice are healthy and show no phenotypic differences from their wild-type littermates [18,18a].Cortical cultures from such mice showed no detectable b-secretase activity and much less Ab peptide Inhibition of Ab generation by lowering the activity of the b-secretase may be beneficial for AD treatment.Therefore,
Correspondence to B.Nawrot, Centre of Molecular and
Macromolecular Studies, Polish Academy of Sciences, Department of
Bioorganic Chemistry, Sienkiewicza 112, Lodz, Poland.
Fax: + 48 42 681 5483, Tel.: + 48 42 681 6970,
E-mail: bnawrot@bio.cbmm.lodz.pl
Abbreviations: Ab, b-amyloid peptide/amyloid b-peptide; AD,
Alzheimer’s disease; AP, alkaline phosphatase; APP, human amyloid
precursor protein; BACE, b-site APP cleaving enzyme; CTE,
constitutive transport element; DMEM, Dulbecco’s modified of Eagle
medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HEK293, human transformed primary embryonal kidney cells;
HEK293sw, HEK293 overexpressing human APP 695 with the
Swedish double mutation; IMR-32, human neuroblastoma cells;
pol III, RNA polymerase III; RNAi, RNA interference;
SiRNA, small interfering RNA.
Dedication: This work is dedicated to Prof.Maciej Wiewiorowski on
the occasion of his 85th birthday.
(Received 15 May 2003, revised 14 July 2003,
accepted 7 August 2003)
Trang 2b-secretase is an excellent target for anti-amyloid
therapeu-tic drug design [19]
Hammerhead ribozymes are the smallest catalytic nucleic
acids able to promote, in a sequence-specific manner, the
cleavage of phosphodiester bonds in RNA.This ability
makes ribozymes useful molecular tools for very selective
degradation of mRNA and inhibition of expression of the
genes of unwanted proteins.The successful selection of
hammerhead ribozymes, active in human cell lines, allows
application of a molecular approach to the design of novel
therapeutics [20–22].Up to now it has been demonstrated
that plasmid encoded ribozymes coupled at their 5¢-ends
with a tRNAValgene sequence are expressed intracellularly
with high efficiency [23].Such ribozymes, when mimicking
the 3¢-immature tRNA molecule, are recognized by the
nuclear protein exportin-t (Xpo-t) and are exported
efficiently from the nucleus to the cytoplasm [24–27]
Conjugation of the 3¢-end of the ribozymes with a
consti-tutive transport element (CTE) sequence, which is an
aptameric sequence for cellular helicases [28–30], leads to
the formation of ribozymes highly active in cellular
experi-ments, independent of the secondary structure of the target
mRNA [31]
The present work demonstrates an application of
endo-genously generated tRNAVal-driven and CTE
helicase-associated hammerhead ribozymes as efficient molecular
tools for the inhibition of the biosynthesis of b-secretase and
for the exclusion of the formation of toxic b-amyloid
peptides in HEK293 cells
Materials and methods
Construction of plasmids for ribozyme expression
Two sites of a b-secretase mRNA (b-site APP cleaving
enzyme, BACE, Gene Bank number: AF190725, start
codon 5¢-AUG456-3¢) were chosen as targets for the
hammerhead ribozymes, namely the 5¢-GUC665-3¢ and
5¢-CUC825-3¢ sequences.Templates containing the
hammer-head catalytic sequence, complementary to the selected
target sites of BACE mRNA, were synthesized chemically
The sequence of the template encoding ribozyme Rz-1
(directed toward cleavage of the GUC665 containing
sequence) is 5¢-CGGTTCGAAACCGGGCACTACAAA
AACCAACTTTGCCCTGCCCCCTGATGAGGCCGA
AAGGCCGAAACTTGCCCCTGGTACCCCGGATAT
CTTTTTTTCTATCGCGTCGACCT-3¢ and the template
encoding Rz-2 (targeted toward CUC825 containing
sequence) is 5¢-CGGTTCGAAACCGGGCACTACAAA
AACCAACTTTCACCCTTCCGCTGATGAGGCCGA
AAGGCCGAAAGGTCCCGGTGGTACCCCGGATA
TCTTTTTTTCTATCGCGTCGACCT-3¢.The ribozyme
templates were PCR amplified with primers P1: 5¢-TTCCC
GGTTCGAAACCGGGCACTAC-3¢ and P2: 5¢-CTGCA
GGTCGACGCGATAGAAAAAAA-3¢.PCR products
were digested with KpnI and Csp45I restriction
endonuc-leases and were ligated with pUC-KE-tRNA-CTE plasmids
that had been digested with both KpnI and Csp45I and
treated with calf intestinal alkaline phosphatase.The
sequencing was carried out on an ABI PRISM instrument
with the PCR primer P7 (5¢-CGCCAGGGTTTTCCCAGT
CACGAC-3¢).Oligonucleotides were synthesized in house
or purchased from ESPEC Custom Oligo Service, Japan Plasmids CTE-Rz-1 and pUC-KE-tRNA-CTE-Rz-2, encoding Rz-1 and Rz-2, respectively, were obtained on a multimilligram scale.Constructs containing inactive versions of Rz-1 and Rz-2 were obtained in the same way by using templates as described above but in which the guanosine nucleotides, marked in bold, had been replaced by adenosine nucleotides
Cell culture, transfection and cell lysis Human transformed primary embryonal kidney cell lines HEK293 and HEK293sw (a kind gift from D.Selkoe, Harvard Medical School, Boston, MA, USA) were cultured
in Dulbecco’s modification of Eagle’s medium (DMEM, Sigma), supplemented with 10% foetal bovine serum (Gibco BRL), 100 lgÆmL)1streptomycin and 100 UÆmL)1 penicillin.G418 (200 lgÆmL)1, Gibco BRL) was used as selection antibiotics for HEK293sw cells.Glass wells were coated with polyL-lysine (Sigma-Aldrich Chemie GmbH) IMR-32 cells were grown in RPMI supplemented with 20% fetal bovine serum and 1% minimal essential medium non essential amino acid (Gibco BRL) and antibiotics as above Glass wells were coated with fibronectin (2 lgÆcm)2, Sigma) Cells were grown in monolayer, at 37C in an atmosphere
of 5% CO2 in 10-cm plates and transfected at 70% confluence.Transfection with 40 lg of the ribozyme-containing plasmid was carried out for 12 h using the LipofectinTMreagent (Gibco BRL) according to the manu-facturer’s protocol.Postincubation was carried out in DMEM for the next 24 h or longer (up to 264 h).Culture medium was collected and frozen at )70 C.Cells were washed three times with Ca+2/Mg+2-free NaCl/Pi and lysed with Tri Pure Isolation Reagent (Boehringer Mann-heim).Lysates were kept at)70 C
Isolation of a total RNA from cell lysates The total RNA fraction was isolated from cell lysates according to the Tri Pure Isolation Reagent protocol (Boehringer Mannheim).The nucleic acid fraction was then treated with RQ1 RNase-free DNase (Promega) and isolated by phenol/chloroform extraction followed
by ethanol precipitation.The total RNA was quantified spectrophotometrically at 260 nm.Samples were kept at )70 C for several months without any decomposition of the RNA
Determination of the level of BACE mRNA and ribozyme RNA expression in mammalian cells The level of BACE mRNA and ribozyme RNA was monitored by RT/PCR using a OneStep RT/PCR kit (Qiagen).For determination of the level of ribozyme RNA the RT/PCR was carried out with two vector primers P1 and P2 (20 lM) and total RNA (0.5 lg).For BACE mRNA level determination the specific BACE gene primers were designed to give a PCR product 430 nucleotides.RT primer (5¢-GCCTTCCCAGTTGGAGCCGTTGAT-3¢, P1BACE), PCR primer (5¢-CGCAGCGGCCTGGGGGGCGCCC C-3¢, P2BACE) and total RNA (0.5 lg) were used for the RT/PCR reaction.PCR was programmed for 30 cycles.The
Trang 3reaction product was analysed by 3% NuSieve GTG
agarose (FMC BioProducts) gel electrophoresis and stained
with ethidium bromide.For experiments performed in
co-amplification conditions two pairs of primers P1BACEand
P2BACE, for BACE mRNA (20 lM), and P1GAPDH and
P2GAPDH, for the control glyceraldehyde-3-phosphate
dehydrogenase (GAPDH; 1.3 lM) were used
Western blotting
HEK293 cells that had been transfected with
ribozyme-expression plasmids were lysed with Tri Pure Isolation
Reagent according to the manufacturer’s protocol.The
protein fraction was isolated and 200 lg sample was
analysed by SDS/PAGE (12% acrylamide).After
electro-phoresis, protein bands were transferred to a
poly(vinylid-ene difluoride) membrane (Millipore).The membrane was
blocked with 5% nonfat milk in NaCl/Tris containing 0.1%
NaN3, probed with primary rabbit polyclonal antibodies
M83 (Santa Cruz) against BACE for 1.5 h at 37C
(dilution 1 : 500) and then with secondary anti-rabbit
polyclonal IgG–alkaline phosphatase (AP; Santa Cruz)
for 1.5 h at room temperature (dilution 1 : 500) Bands
were visualized by addition of Nitro Blue tetrazolium and
5-bromo-4-chloroindol-3-yl phosphate (Sigma-Aldrich
Chemie GmbH) in 100 mMTris/HCl pH 9.5 buffer
con-taining 100 mMNaCl and 5 nMMgCl2
Immunoprecipitation of b-amyloid peptide
and dot blot analysis
The level of b-amyloid peptide in conditioned cultured
medium and in cells was determined by
immunoprecipita-tion and dot blot analysis.Condiimmunoprecipita-tioned medium was
collected from the ribozyme-transfected HEK293 cell
cul-ture postincubated for 48 or 60 h.Conditioned medium
from cells transfected with lipofectin only was used as a
control.The conditioned medium (1 mL) was subjected to a
preclearing process by treatment with Protein A Sepharose
4 Fast Flow (50 lL, 50% slurry, Amersham Pharmacia
Biotech AB).Immunoprecipitation of the b-amyloid
pep-tide Ab40 with primary rabbit polyclonal antibody
Anti-b-Amyloid 40 (BioSource International Inc.) was performed
according to the Immunoprecipitation Starter Pack
proto-col (Amersham, Pharmacia Biotech AB).For antigene–
antibody complex precipitation Protein A Sepharose 4 Fast
Flow was added (50 lL, 50% slurry) to the precleared
conditioned medium (500 lL).The complex was washed
three times with lysis buffer (NP-40) and once with wash
buffer (50 mMTris, pH 8.0) The pellet was suspended in
reducing buffer (1% SDS, 100 mM dithiothreitol, 50 mM
Tris pH 7.5), heated for 3 min at 95C and centrifuged at
12 000 g for 20 s.Supernatant (5 lL) was applied to a
cellulosenitrate (E) membrane (Schleicher & Schuell).The
membrane was dried, washed in NaCl/Tris and incubated in
5% nonfat milk for 1.5 h at room temperature After
saturation the membrane was washed twice with NaCl/Tris
and then treated with the secondary anti-rabbit IgG
conjugated with AP (Santa Cruz Biotechnology) for 1.5 h
at room temperature.After three washes in NaCl/Tris
supplemented with 0.04% Tween 20, and once with AP
buffer (100 m Tris pH 9.5, 100 m NaCl), the membrane
was incubated with Nitro Blue tetrazolium and 5-bromo-4-chloroindol-3-yl phosphate and washed finally with ddH2O Immunoprecipitation of b-amyloid peptide Ab40 from the ribozyme-transfected HEK293 cells postincubated for
48 or 60 h was performed as follows.Cultured cells were lysed with Tri Pure Isolation Reagent according to the manufacturer’s protocol.The preclearing process of the cell protein extract (100 lg of protein in 500 lL of extract) was carried out as described above.Immuno-precipitation and determination of the intracellular level
of Ab40 was carried out according to the procedure described above
Synthetic b-amyloid peptide, fragment 1–40 (Sigma-Aldrich Chemie GmbH) (100 ng) was used as a control for the dot blotting process and for quantification of the immunoprecipitation level.The blots were quantified by using theIMAGE QUANTprogram
Results
Design of ribozymes Ribozymes with a hammerhead catalytic core possess the ability to cleave substrate RNA after the 5¢-NHH-3¢ sequence, where N is A, U, C or G and H is A, U or C [32], with the most efficient cleavage of the substrate possessing the triplet GUC or CUC [23].The messenger RNA of BACE, which functions as a b-secretase in the process of releasing Ab, was selected as our target molecule Two sites of the BACE mRNA were chosen for hammer-head cleavage: 5¢-GUC665-3¢ and 5¢-CUC825-3¢.The lowest energy secondary structure of BACE mRNA, generated with the help of theMFOLD program [33] shows that the 5¢-GUC665-3¢ target sequence is not involved in hydrogen bonding.However, the availability of the 5¢-CUC825-3¢ triplet, which is the target sequence for Rz-2, as well as the availability of the recognition arms of both substrates, are rather poor for hybridization with ribozymes.To overcome the problem of target/ribozyme involvement within the intramolecular hydrogen bonding, we took advantage of our recent achievements in the design of ribozymes with significantly improved intracellular activity [23–26,31] First, we applied tRNAVal-driven ribozymes.An intra-cellular transcription of such tRNAVal-driven ribozymes is performed by the RNA polymerase III (pol III) system and provides a high level of endogenous ribozyme expression Moreover, in somatic cells, the transport of tRNA-mimicking transcripts of ribozymes from nucleus to cyto-plasm is facilitated by Xpo-t [27].Such ribozymes exhibit significantly higher cytoplasmic activity due to their colocal-ization with the target mRNA.The tRNA-driven ribozymes were flanked on their 5¢-ends by a tRNAValmotif [25].The last seven bases of the wild-typemature tRNAValwere replacedby
a linker 5¢-ACUACAAAAACCAAC-3¢ sequence, which prevents the processing of the 3¢-end of the transcript and secures cloverleaf secondary structure of the tRNA motif Extension of the linker by the three additional uridine nucleotides allowed us to design ribozymes with the required secondary structure, as generated byMFOLD
To ensure the efficacy of endogenously generated cata-lytic nucleic acids our hammerhead ribozymes were exten-ded at their 3¢-termini with a CTE sequence.The CTE motif
Trang 4was identified as the aptameric RNA sequence for cellular
RNA helicases [28–30].It has already been shown that
attachment of a CTE sequence to the ribozyme cassette
results in significant improvement of ribozyme efficacy [31]
The intracellular helicase-associated ribozyme possesses an
ability to unwind double-stranded sequences of the
mes-senger RNA and facilitates the change of mechanism by
which the ribozyme searches for its target site.With such a
hybrid sequence the ribozyme reaches its target site by the
sliding mechanism used by cellular helicases.This
pheno-menon significantly improves the efficacy of protein-hybrid
ribozymes, due to the ability of such complexes to guide the
ribozyme to its target site
Expression of ribozyme plasmids in mammalian cells
Three cultured cell lines HEK293, HEK293sw and IMR-32
were used to study the expression of ribozymes, which were
cloned into pUC-KE-tRNA-CTE plasmids.Ribozyme
encoding plasmids were transiently transfected into the
tested cells with the aid of lipofectin.Expression of the
ribozymes was observed in the course of time.The nucleic
acid fraction, isolated from cell lysates, was treated with
RNase-free DNase I to remove traces of plasmid DNA,
and the total RNA was used as a reverse transcription
template for semiquantitative RT/PCR analysis of the level
of ribozyme RNA in the cells.Amplification of
pUC-KE-tRNA-CTE-Rz-1 and Rz-2 plasmids gave an expected
295-bp product (Fig.1, lanes 2 and 4, respectively).When RT/
PCR was carried out with an exclusion of the reverse
transcription step, the reaction did not yield the
corres-ponding amplification product (lanes 1 and 3).This result confirmed that the RT/PCR product of the expected length did originate from the cellular RNA fraction, and not from plasmid DNA amplification
Similar results were obtained while monitoring an expression of ribozyme plasmids pUC-KE-tRNA-CTE-Rz-1 and Rz-2 in IMR-32 and HEK293sw cells, although the level of ribozyme RNA present in the lysates was significantly lower ( 60% and 20%, respectively, data not shown).This could be caused by decreased transfection efficiency and/or by a low ribozyme expression efficiency in the genetically changed HEK293sw cells
The expression of pUC-KE-tRNA-CTE-Rz-1 in HEK293 cells was also observed over a longer time interval (Fig.2) As expected for transient transfection of the plasmid, the expression of ribozyme reached its maximum after 24 h of postincubation.The level of ribozyme signi-ficantly decreased during the following 24 h, but even after
11 days we could still detect traces of ribozyme RNA (data not shown)
Activity of ribozymes in HEK293 cells
At first we checked the activity of the ribozymes by determining the level of the target mRNA in tested cells transfected with ribozyme encoding plasmids.Two sites
of BACE mRNA, 5¢-GUC665-3¢ and 5¢-CUC825-3¢, were selected as target sequences for ribozymes Rz-1 and Rz-2, respectively.Specific primers for the RT/PCR (P1BACEand P2BACE) were designed in such a way that ribozyme degradation sites were included within the amplified BACE mRNA sequence.The amplification product was expected
to be 430 bp DNA.As the expression of b-secretase in HEK293 cells is rather low [12,13] it was necessary to determine the number of the PCR cycles suitable for monitoring the changes of the BACE mRNA level.Under standard RT/PCR conditions (0.5 lg total RNA and 20 lM primers) it was possible to observe the appearance of the
Fig 1 Expression of pUC-KE-tRNA-CTE-Rz-1 and Rz-2 plasmids
in HEK293 cells determined by semiquantitative RT/PCR Ribozyme
encoding plasmids were transiently transfected into the cells.Thirty-six
hours after transfection, the nucleic acid fraction was isolated from cell
lysates, treated with RNase-free DNase I and used in RT/PCR.The
reaction products were analysed by electrophoresis through 3%
agarose and stained with ethidium bromide.As expected, 295-bp
products of reverse transcription and PCR amplification of Rz-1 and
Rz-2 plasmids with specific vector primers P1 and P2 (see Materials
and methods) were obtained (lanes 2 and 4, respectively).When RT/
PCR was performed without a reverse transcription step, the reaction
did not yield the corresponding amplification product (lanes 1 and 3).
Fig 2 Semi-quantitative analysis of expression of the pUC-KE-tRNA-CTE-Rz-1 plasmid in HEK293 cells over time After transfection with Rz-1 plasmid, HEK293 cells were lysed 0, 6, 12, 24, 36 and 48 h post incubation.The total RNA fraction was isolated from the cell lysates, treated with RNase-free DNase I and used in RT/PCR.At postincu-bation time 0 h (12 h after the beginning of transfection, see Materials and methods) only a minimal amount of ribozyme RNA was present
in the cell lysates.The level of ribozyme increased with time and reached its maximum 24 h after transfection.The expression of GAPDH mRNA was monitored as internal control.
Trang 5430-bp product only after more than 25 PCR cycles.With
more than 30 PCR cycles the amount of amplification
product reached a plateau.Thus, in our RT/PCR protocol
we used 30 PCR cycles as the standard.The mRNA of the
GAPDH protein was amplified as a control.Primers for
amplification of GAPDH (P1GAPDHand P2GAPDH) were
designed to give a 150-bp product.In co-amplification
RT/PCR conditions the concentration of primers P1GAPDH
and P2GAPDHwas 15 times lower than primers P1BACE
and P2BACE
The HEK293 cells were transfected with plasmids
encoding Rz-1, Rz-2 and with their inactive versions
(Rz-1i and Rz-2i).Transfections of the cells with lipofectin
(NO) only or lipofectin and an empty plasmid (EM) were
used as controls.The level of the RT/PCR products was
determined under co-amplification conditions 36 h after
transfection.Figure 3 shows an agarose gel electrophoresis
of the RT/PCR products from different transfection
experiments.While the level of the 150-bp control product
is constant for all six experiments (and below the saturation
of the PCR reaction) we could observe no or very little
BACE mRNA amplification product (430 bp) for
experi-ments where the Rz-1 and Rz-2 plasmids were used
However, no significant lowering of the BACE
amplifica-tion product was observed for experiments where inactive
forms of ribozymes were used
The level of BACE mRNA in Rz-1 transfected HEK293
cells was also monitored in the course of time under single
gene amplification RT/PCR conditions (Fig.4).Cells were
lysed after 6, 12, 24, 36 and 48 h of the postincubation time
The level of GAPDH mRNA was determined in parallel
separate experiments.The amount of amplification product
decreased for the first 24 h after transfection, when it
reached < 5% of the control BACE mRNA expressed in
the cells transfected with lipofectin only (NO) and lysed
after 48 h.Over the next several hours we observed an
increase of the BACE mRNA level because of the lower expression of ribozyme.A similar profile of the target mRNA was obtained for the cells transfected with the Rz-2 plasmid (data not shown)
Effect of ribozyme expression on BACE protein and b-amyloid peptide levels
To examine whether transiently expressed ribozymes affect the amount of b-secretase gene expression on the protein level, Western blotting analyses were performed.The test cells were transfected with Rz-1 and Rz-2 encoding plasmids and after 36 h of postincubation the protein fraction was isolated from the cells and subjected to analysis.As shown
in Fig.5 the extent of protein expression is much lower in the HEK293 cells treated with Rz-1 ( 5%) and with Rz-2 ( 10%) plasmids in comparison to the amount of BACE
in the control transfected cells.A protein mixture of known molecular mass was used as a marker to establish the band
of BACE protein with a molecular mass of 70 kDa
As our ribozyme constructs were active in the cellular system and efficiently inhibited biosynthesis of b-secretase
Fig 3 Expression of BACE and GAPDH mRNA in HEK293 cells
transfected with ribozyme plasmids monitored by RT/PCR Plasmids
encoding Rz-1, Rz-2 and their inactive versions Rz-1i and Rz-2i were
used for cell transfection.As controls, only lipofectin (NO) or
lipo-fectin and an empty plasmid (EM) were used.The level of the RT/PCR
products was determined under co-amplification conditions 36 h after
transfection.An agarose gel electrophoresis of the RT/PCR products
demonstrates that the level of the 150-bp control product is constant
for all six experiments, while no or very little BACE mRNA
amplifi-cation product (430 bp) is observed for experiments where the Rz-1
and Rz-2 plasmids were used.
Fig 4 The level of BACE mRNA in HEK293 cells transfected with pUC-KE-tRNA-CTE-Rz-1 monitored over time under noncompetitive RT/PCR conditions Cells were lysed 6, 12, 24, 36 and 48 h post incubation.The level of GAPDH mRNA was determined in parallel separate experiments.The amount of amplification product decreased for the first 24 h after transfection, when it reached less than 5% of the control BACE mRNA expression in the cells transfected with lipo-fectin only (NO) and lysed after 48 h.
Fig 5 Effect of ribozyme expression on BACE protein level in HEK293 cells The test cells were transfected with Rz-1 and Rz-2 encoding plasmids and 36 h postincubation the protein fraction was isolated from the cells and subjected to Western blotting analysis.The extent of protein expression is much lower in the HEK293 cells treated with Rz-1 ( 5%) and with Rz-2 ( 10%) plasmids relative to the amount
of BACE in the control transfected cells (NO, EM, Rz-1i and Rz-2i).
Trang 6we asked the question whether we could observe any
difference in the level of extra- and intracellular release of
b-amyloid peptide, which is a product of the catalytic
activity of the target protein.Our efforts to use Western blot
analysis for detection of the presence of 4-kDa b-amyloid
peptide in the cell extracts, or in the conditioned cultured
medium, even using as much as 300 lg of the protein
fraction, were unsuccessful.This was probably because of
the very low level of b-amyloid peptide in the test cells [2,3]
Thus, we performed immunoprecipitation of the b-amyloid
peptide Ab40 either from cell lysates or from conditioned
cultured medium.The immunoprecipitation reaction was
performed with primary rabbit polyclonal antibody
(Anti-b-Amyloid 40).Detection of the Ab/Ab40 complex was
per-formed by treatment of the membrane with the secondary
anti-(rabbit IgG AP) Ig followed by dot blot visualization
with Nitro Blue tetrazolium and
5-bromo-4-chloro-indol-3-yl phosphate reagents.A synthetic b-amyloid
peptide, fragment 1–40, was used as a control in
immuno-precipitation experiments
In cell cultures transfected with our ribozyme constructs
the level of extra- and intracellular Ab peptide was
significantly decreased.Figure 6 shows the data for the
extracellular level of the Ab peptide 48 and 60 h after
transfection of the HEK293 cells with the Rz-1 and Rz-2
encoding plasmids.Forty-eight hours after transfection
the amount of Ab peptide reached the level of 65% and
40%, respectively, for cells transfected with Rz-1 and
Rz-2, in comparison to the level of a control experiment
with lipofectin only transfected cells.A further decrease of
the Ab level up to 20% was observed in the following
12 h.However, the intracellular level of Ab peptide
decreased only up to 60% during 60 h after transfection
with Rz-1 and Rz-2 plasmids in comparison to the control
lipofectin only transfection (data not shown)
Discussion
Engineered ribozymes are of great interest as modern therapeutic agents due to their potential to specifically and efficiently inhibit either unwanted proteins or viral RNA gene expression via catalytic hydrolysis of specific internu-cleotide bonds of the target RNA.The functional activity of endogenously delivered ribozymes depends on many factors including their intracellular concentration, an effective export from the nucleus to the cytoplasm, a colocalization
of the ribozyme with the target RNA and the availability of the target sites for enzymatic cleavage.Computer-generated secondary structures of long messenger RNAs suggest their poor availability for association with complementary strands, such as ribozyme and antisense oligonucleotides
We have used tRNAVal-driven CTE-conjugated hammer-head ribozyme cassettes, recently designed by one of our laboratories [24–27,31].These engineered hammerhead ribozymes fulfil the requirements to be active in a cellular system independently of the secondary structure of the target mRNA
As the target molecule we have chosen the gene for human b-secretase, which is an aspartyl protease Asp2, also called BACE protein or memapsin 2 [9–13].This protein is involved in proteolytic cleavage of an amyloid precursor protein APP, and directly influences the release of the toxic b-amyloid peptide.Ab is thought to be a primary patho-genic agent in AD [1].It was found as a main component of the aggregates leading to ageing dementia
There are many approaches to reduce the level of the b-amyloid peptide.It was thought that inhibition of expression of APP by antisense oligonucleotides might be
of interest for therapeutic application [34].However, because of the functional importance of nonamyloidogenic APP secretion products in healthy individuals this approach does not seem to be suitable.Another approach to the reduction of Ab formation utilizes an application of noncleavable analogues of BACE substrates [14–17].Several differently modified short peptides were chemically synthes-ized and used successfully for inhibition of b-secretase activity.However, there are several other aspartyl proteases present in cells [19].Some of them, e.g.cathepsin D, show quite remarkable affinity for BACE inhibitors [14].Thus, there is still a need for novel molecular tools which would be able to effectively reduce the level of BACE protein, and in consequence, to reduce the amount of Ab peptide released in neuronal cells
We asked the question whether we can inhibit expres-sion of the gene of BACE protein using our engineered ribozymes.In order to check the potential of plasmid coded ribozymes for cleavage of the target sequence we performed an in vitro cleavage reaction of the short 5¢-radioactively labelled RNA substrates (25-mers) with ribozymes obtained by an in vitro transcription.As expected, both ribozymes transcripts exhibit potential for cleaving their short complementary RNA targets (data not shown)
Although the highest level of expression of BACE protein was identified in the neuronal cells of the brain, for preliminary experiments we have chosen human embryonal kidney (HEK293) cells as well as HEK293sw cells overex-pressing APP with a Swedish mutation and IMR-32 human
Fig 6 Effect of Rz-1 and Rz-2 activity on the extracellular level of
b-amyloid peptide b-Amyloid peptide Ab40 was immunoprecipitated
from the conditioned cultured medium collected 48 and 60 h after
transfection of HEK293 cells with the test ribozyme
plasmids.Forty-eight hours after transfection, the amount of Ab peptide reached the
level of 65% and 40%, respectively, for cells transfected with
Rz-1 and Rz-2, relative to the level of a control experiment with
lipofectin only transfected cells (first three bars).Further decrease of
the Ab level, to below 20%, was observed in the following 12 h
(second three bars).
Trang 7neuroblastoma cells.Ribozyme cassettes were introduced
into the bacterial plasmid pUC-KE and used for
transfec-tion of the test cells.Intracellularly generated ribozyme
transcripts were identified in the cell extracts, as shown in
Figs 1 and 2.Our goal was to downregulate endogenous
BACE mRNA and the target protein as well as to prevent
b-amyloid peptide formation.We were able to demonstrate
by semiquantitative RT/PCR that an increasing amount of
expressed ribozyme directly influences the intracellular level
of endogenous BACE mRNA (Fig.7).The maximum level
of ribozyme expression occurs 24 h after transfection, while
after that time the expression of BACE mRNA reaches a
minimum ( 5%).Typically, for transient transfection the
level of expressed ribozyme drops down; however, it could
be still detected by RT/PCR even 11 days after transfection
Expression of ribozymes in the other two test cell lines
was limited to 60% and 20%, respectively, for the IMR-32
and HEK293sw cells.This could be caused by the different
efficiency of the plasmids in the transfection of the test cells
or by low ribozyme expression efficiency in the genetically
changed HEK293sw cells.As a consequence, the low level
of ribozyme expression significantly reduced the efficiency
of the BACE mRNA degradation (data not shown)
Probably a much bigger amount of ribozyme plasmids
should be used for transfection of HEK293sw and IMR-32
cells to reach satisfactory inhibition of BACE expression
The decreased amount of BACE mRNA in HEK293
cells led to the lower biosynthesis of this protein (up
to 90%), as monitored 36 h after transfection (Fig.5)
Lowering of BACE amount should directly result in
decreased amount of APP cleavage products (b-amyloid
peptide, N-terminal b-APPs and b-CTF
fragment).Immuno-precipitation of Ab from cell lysates as well as from cell
culture medium 48 and 60 h after transfection showed
significantly decreased amounts of both the intra- and
extracellular b-amyloid peptide in comparison to the Ab isolated from control lipofectin only transfected cells Functional effects of hammerhead ribozymes as well as their inactive forms were not tested for their influence on the level of other APP proteolysis products.However, as reported earlier, lowering of BACE expression results in lowering of the released amount of Ab, b-APPs and b-CTF fragments [9,11,18]
Although the downregulation of BACE is only partial and transient, it is necessary to test whether our ribozyme constructs are effective for BACE gene silencing and reduction of the level of the toxic b-amyloid peptides in neuronal cell lines.Further experiments on APP transgenic mouse, exhibiting an elevated level of b-amyloid peptide [35], are needed to demonstrate in vivo activity of such ribozymes.The tRNAVal-driven and CTE-coniugated ribo-zymes directed toward murine BACE mRNA are under preparation in our laboratories.Our results kindle some hopes that such hammerhead ribozymes may be used as molecular tools for the specific inhibition of b-secretase activity
Recently, there is growing interest in RNA interference (RNAi) phenomena [36,37].The power of RNAi is remarkable, because it can serve as a very efficient sequence-specific tool for gene silencing.RNAi is a valuable therapeutic tool for drug design and operates in the cytoplasm.It is induced by small interfering RNAs (siRNAs), which are the products of a double-stranded RNA cleavage by a nuclease dicer [38].Selective inactivation
of genes for BACE1 and BACE2 by siRNA was reported recently [39].Up to now several expression systems have been designed for endogenous generation of siRNA [40–44]
As our tRNA-driven ribozyme transcripts are efficiently exported from the nucleus to the cytoplasm, where they meet their target mRNA, this expression system is of interest for endogenous generation of siRNA and activation of the RNAi effect in human cells [45].Studies on an inhibition
of BACE gene expression by endogenously generated siRNA via tRNAVal promotion are in progress in our laboratories
Acknowledgements
This work was supported by Polish Committee for Scientific Research (project PBZ-KBN-059/T09/09) and European Commission (5th Framework Programme, NAS Complement to the project QLK6-CT-1999–02112).We thank Wieslawa Goss and Barbara Mikolajczyk for excellent technical assistance.
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