Correction of Alternative Splicing of Tau in Frontotemporal

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Correction of Alternative Splicing of Tau in Frontotemporal Powered By Docstoc
					THE JOURNAL   OF   BIOLOGICAL CHEMISTRY                                                        Vol. 276, No. 46, Issue of November 16, pp. 42986 –42993, 2001
                                                                                                                                             Printed in U.S.A.



Correction of Alternative Splicing of Tau in Frontotemporal
Dementia and Parkinsonism Linked to Chromosome 17*
                                                           Received for publication, June 4, 2001, and in revised form, September 11, 2001
                                                        Published, JBC Papers in Press, September 17, 2001, DOI 10.1074/jbc.M105113200


                   Bernd Kalbfuss, Stephen A. Mabon, and Tom Misteli‡
                   From National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892



   Mutations in the human tau gene cause frontotempo-                       Apart from FTDP-17, tau deposits are also the defining path-
ral dementia and Parkinsonism associated with chromo-                       ological feature of several neurological disorders including
some 17 (FTDP-17). One of the major disease mecha-                          Alzheimer’s disease, progressive supranuclear palsy, and
nisms in FTDP-17 is the increased inclusion of tau exon                     Niemann-Pick disease (4, 6).
10 during pre-mRNA splicing. Here we show that modi-                           Tau is a microtubule-binding protein that normally promotes
fied oligonucleotides directed against the tau exon 10                      microtubule assembly and stability (12–15). The microtubule-
splice junctions suppress inclusion of tau exon 10. The                     binding domains are encoded by exons 9 –12 (16). In human
effect is mediated by the formation of a stable                             brain, six tau isoforms are expressed as the result of alterna-
pre-mRNA-oligonucleotide hybrid, which blocks access                        tive use of exon 2, exon 3, and exon 10, which encodes one of the
of the splicing machinery to the pre-mRNA. Correction
                                                                            microtubule-binding domains (17). Increased inclusion of exon
of tau splicing occurs in a tau minigene system and in




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                                                                            10 results in an elevated level of tau protein containing an
endogenous tau RNA in neuronal pheochromocytoma
                                                                            additional fourth microtubule-binding domain (the 4R isoform)
cells and is specific to exon 10 of the tau gene. Antisense
oligonucleotide-mediated exclusion of exon 10 has a                         relative to tau protein containing only three microtubule-bind-
physiological effect by increasing the ratio of protein                     ing domains (the 3R isoform). Increased inclusion of tau exon
lacking the microtubule-binding domain encoded by exon                      10 is tightly correlated with the disease phenotype, and over-
10. As a consequence, the microtubule cytoskeleton be-                      production of 4R tau is sufficient for disease (10, 18). The
comes destabilized and cell morphology is altered. Our                      increase in exon 10 inclusion is due to mutations at the 3 -end
results demonstrate that alternative splicing defects of                    of exon 10, which result in the destabilization of an RNA
tau as found in FTDP-17 patients can be corrected by                        stem-loop (19). In healthy individuals, this loop prevents access
application of antisense oligonucleotides. These findings                   of the splicing machinery to exon 10, thus blocking its inclusion
provide a tool to study specific tau isoforms in vivo and                   into the mRNA. When mutated, the stem-loop is destabilized,
might lead to a novel therapeutic strategy for FTDP-17.                     and the splice junction in exon 10 can be used (4, 19 –23). In
                                                                            addition, cis-acting elements within exon 10 also contribute to
                                                                            splice site choice (23, 24).
   Alternative splicing is a major regulatory mechanism in gene                Here we report the application of antisense oligonucleotides
expression. An estimated 40% of human genes are alterna-                    to reverse the inclusion of tau exon 10 into mRNA as observed
tively spliced, and about 15% of the reported mutations in                  in FTDP-17 patients. Modified antisense oligonucleotides di-
human genetic diseases affect pre-mRNA splicing (1–3). One of               rected against either splice junction of exon 10 efficiently cor-
the diseases directly caused by mutations that result in                    rected its inclusion into tau mRNA. The oligonucleotides elic-
pre-mRNA splicing defects is frontotemporal dementia and                    ited a physiological effect by reducing the level of tau protein
Parkinsonism associated with chromosome 17 (FTDP-17)1 (4 –                  containing the microtubule-binding domain encoded by exon
6). FTDP-17 is an autosomal-dominant disorder related to Alz-               10, and as a consequence the cytoskeleton morphology was
heimer’s disease. Symptoms of FTDP-17 include personality                   altered. The use of antisense oligonucleotides will be useful to
changes, reduced speech, and dementia. In late stages, memory               study the physiological role of specific tau isoforms in disease
loss occurs, and the disease becomes virtually indistinguish-               and cellular differentiation, and it might provide a novel ther-
able from Alzheimer’s disease (7). The disease gene for                     apeutic strategy to treat FTDP-17 and other tauopathies.
FTDP-17 has been identified by positional cloning as the tau
gene, which encodes a microtubule-binding protein (8, 9). In                                   EXPERIMENTAL PROCEDURES
FTDP-17, tau is incorporated into aggregates of fibrillar tan-                 Cell Lines—COS-1 green monkey kidney cells (ATCC entry CRL-
gles and paired helical filaments, leading to neuronal cell death           1650) were grown in Dulbecco’s modified Eagle’s medium plus 10% fetal
causing the neurological symptoms of the disease (4, 6, 10, 11).            bovine serum at 37 °C. Rat PC12 cells (ATCC entry CRL-1721) were
                                                                            grown in RPMI 1640 medium, 10% fetal bovine serum, 5% horse serum,
                                                                            2.5 g/liter glucose, 1 mM sodium pyruvate. Cells were split every other
                                                                            day at a ratio of about 2:3. A Pasteur pipette was used to deaggregate
  * The costs of publication of this article were defrayed in part by the   cell clusters. Rat AR42J pancreatic acinar cells (ATCC entry CRL-1492)
payment of page charges. This article must therefore be hereby marked       were grown in F-12/Ham medium, 10% fetal calf serum.
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
                                                                               Modified Antisense Oligonucleotides—All oligonucleotides were 2 -O-
indicate this fact.
  ‡ To whom correspondence should be addressed: NCI, National Insti-        methyl-oligoribonucleotides, contained a phosphorothioate backbone,
tutes of Health, 41 Library Dr., Bldg. 41, Bethesda, MD 20892. Tel.:        and were purified by high pressure liquid chromatography. Identical
301-402-3959; Fax: 520-832-0970; E-mail: mistelit@mail.nih.gov.             results were obtained with oligonucleotides from either Operon Tech-
  1
    The abbreviations used are: FTDP-17, frontotemporal dementia and        nologies or from Hybridon: E10 , 5 -UAUCUGCACCUUUGGUAG-3 ;
Parkinsonism associated with chromosome 17; PCR, polymerase chain           E10 , 5 -UGAAGGUACUCACACUGCCGC-3 ; rat E10 , 5 -CGACAG-
reaction; RT, reverse transcriptase; bp, base pair(s); PIPES, 1,4-pipera-   UACUCACACUGCCUC-3 .
zinediethanesulfonic acid; PBS, phosphate-buffered saline; rE10 , rat          Transfection—Twenty hours prior to the experiment, 1.2 106 cells
E10 .                                                                       were plated on a 60-mm Petri dish. For co-transfection of the tau

                                                                      42986                         This paper is available on line at http://www.jbc.org
                                                         Correction of Tau Splicing                                                         42987
minigene and oligonucleotides into COS-1 cells, GenePorter lipofection       2% milk, 0.1% Tween) for 60 min at room temperature. The signal was
reagent was used according to the manufacturer’s protocol using a            visualized using the Amersham ECL or ECL plus Western blotting
DNA/GenePorter ratio of 1:3. Typically, 21 l of GenePorter reagent in        detection kits and Hyperfilm ECL (Amersham Pharmacia Biotech).
2 ml of serum-free Dulbecco’s modified Eagle’s medium were used per          Bands were quantitated on a FluorChem system (Alpha Innotech).
sample. 2 ml of 20% fetal bovine serum medium were added 5 h later.             Fluorescence Microscopy—A42J cells were grown on coverslips and
For transfection of PC12 cells, electroporation was used. Electropora-       transfected as described for COS-1 cells. 48 h after transfection, cells
tion resulted in significantly higher efficiency of oligonucleotide uptake   were fixed for 12 min in fresh 3.7% paraformaldehyde in CSK buffer (10
as assayed by monitoring uptake of fluorescently labeled oligonucleo-        mM PIPES, pH 6.8, 100 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 1 mM
tides. For electroporation, a single-cell suspension was made by resus-      phenylmethylsulfonyl fluoride, 0.1% Triton X-100). Cells were rinsed in
pending cells in 2 ml of Dulbecco’s modified Eagle’s medium and forcing      PBS and incubated for 60 min with anti-tubulin antibody (Sigma),
them several times through a 25 5⁄8-inch syringe needle. 0.4 ml of cell      1:500 in PBS. Coverslips were then rinsed three times for 8 min each in
suspension in serum-free medium at 6           106 cells/ml was electropo-   PBS and incubated for 60 min with rabbit anti-mouse fluorescein iso-
rated in a 2-mm gap electroporation cuvette (Bio-Rad) using a BTX            thiocyanate (Vector Laboratories), 1:250 in PBS. Cells were rinsed
electroporator (five pulses, 150 V, 4 ms, 3-s interval). DNA concentra-      three times for 8 min each and mounted in Vectashield (Vector Labo-
tion was adjusted to 20 g with salmon sperm carrier DNA. Samples             ratories). Fluorescence microscopy was performed on a Leica TCS-SP
were cultured in about 3.5 ml of serum-free medium supplemented with         confocal microscope using a 100, 1.4 NA objective. Excitation was at
10% fetal bovine serum and 5% horse serum afterward.                         488 nm, and emission was detected between 525 and 575 nm. Single
   RNA Extraction—RNA was extracted using the RNAWiz phenol                  optical sections of 500-nm nominal thickness were collected. Phase
extraction kit (Ambion) according to the manufacturer’s instructions.        images were acquired on a Nikon Eclipse 800 microscope equipped with
Cells were scraped from plates 20 h post-transfection in 1 ml of cold        a MicroMax cooled CCD camera and Metamorph Imaging software.
PBS and spun down for 4 min at 2000               g at 4 °C. Pellets were    Raw images are shown.
resuspended in 500 l of RNAWiz. Precipitated and washed RNA pel-
lets were resuspended in diethyl pyrocarbonate-treated water, and                                           RESULTS
concentrations were measured using a spectrophotometer.                         We sought to correct inclusion of exon 10 into tau mRNA by
   RT-PCR—RNA was treated with 1 unit of RQ1 DNase for 30 min,
                                                                             use of antisense oligonucleotides. Our approach was to intro-
and DNase was heat-inactivated at 95 °C for 5 min. PerkinElmer Life
                                                                             duce into cells 2 -O-methyl-oligoribonucleotides containing se-




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Sciences murine leukemia virus reverse transcriptase and oligo(dT)
18-mer first strand primers (Life Technologies, Inc.) were used for          quence complementary to the 3 or 5 splice site of exon 10 (Fig.
reverse transcription. The reverse transcription reaction was set up to      1, a and c). In contrast to unmodified phosphorothioate oligode-
contain 1–5 g of RNA in 0.5 mM dNTP (Life Technologies), 5 M                 oxynucleotides, 2 -O-methyl-modified antisense oligonucleo-
oligo(dT)18 (Life Technologies), 5 mM MgCl2 in a 16- l volume. The           tides do not promote RNase H-mediated RNA degradation but
reaction was preincubated for 3 min at 80 °C, cooled to 42 °C, and spun
                                                                             rather form stable RNA-DNA hybrids (26). Our rationale was
down. 10 PCR buffer, 20 units of RNase Inhibitor (PerkinElmer Life
Sciences), and 50 units of murine leukemia virus reverse transcriptase       that the oligonucleotides prevent access of the splicing machinery
were added to give a volume of 20 l. The reaction was incubated for 60       to the splice junction and in this way may block the inclusion of
min at 42 °C. Reverse transcriptase was heat-inactivated at 92 °C for 10     the protected tau exon 10 into the mRNA (Fig. 1, a and c).
min. For the PCR reaction, 5 l of the RT reaction, 200 M dNTP, 1 M              Exclusion of Tau Exon 10 in a Minigene System—To test
of each primer, 10 Pfu buffer, and 2.5 units of Pfu Turbo (Stratagene)       whether antisense oligonucleotides targeted to tau exon 10
were used. PCR was performed (1 min at 94 °C; 36 cycles of 1 min at
                                                                             sequences were capable of altering the splicing behavior of tau
94 °C; 1 min at 60 °C; 1 min at 72 °C; 10 min at 72 °C) on a Hybaid
Express thermal cycler. Primers for amplification of tau minigne con-        exon 10, we used a previously characterized minigene system
structs were located in the TAT splice donor and acceptor of pSBL3b of       that recapitulates to a large extent the behavior of exon 10 in
the Exon Trapping System (Life Technologies). The primers were SD2           the context of the full-length tau gene (21). The splicing pattern
(5 -GTGAACTGCACTGTGACAAGC-3 ) and SA4 (5 -CACCTGAG-                          of exon 10 in the minigene was analyzed by RT-PCR on total
GAGTGAATTGGTCG-3 ), and the lengths of amplified products were               RNA isolated 24 h after transfection of the minigene into
246 bp (10 ) and 153 bp (10 ). For amplification of endogenous tau
                                                                             COS-1 cells (Fig. 1b). As expected, expression of the wild-type
from PC12 cells or AR42J cells, primers tauex9 (5 -CTGAAGCAC-
CAGCCAGGAGG-3 ) and tauex13 (5 -TGGTCTGTCTTGGCTTTGGC-                        exon 10 minigene in COS-1 cells resulted in predominant ex-
3 ) located in exon 9 and 13, as described by Grover et al. (21), were       clusion of exon 10 (Fig. 1b) (21). The fraction of the 10 tau
used. The length of the amplified products was 367 bp (10 ) and 297 bp       isoform was 0.27       0.08 (Fig. 1b). In contrast, similar to the
(10 ). PCR conditions were as follows: 10 min at 95 °C; 42 cycles of 30 s    situation in FTDP-17 patients, expression of exon 10 contain-
at 95 °C; 40 s at 60 °C; 1 min at 72 °C; 10 min at 72 °C. For probing of     ing a single point mutation at position 1 resulted in predom-
splicing of exons 1 and 4, primers tauex1 (5 -CCC GCC AGG AGT TTG
                                                                             inant inclusion of exon 10 (21). The fraction of the 10 tau
ACA CAAT) and tauex4 (5 -GGC CAC TCG AGC TTG ACTCA) were
used. PCR conditions were as follows: 10 min at 95 °C; 42 cycles of 30 s     isoform was now increased to 0.87          0.06 (Fig. 1b). Similar
at 95 °C, 45 s at 50 °C, and 60 s at 72 °C; 10 min at 72 °C. The length      results were obtained with mutants at positions 3 and 14
of the amplified products was 365 nucleotides for the isoform including      (data not shown; Ref. 21). The identity of the PCR products was
exons 2 and 3, 268 nucleotides for the isoform including exon 2 only, and    confirmed by sequencing (data not shown).
180 nucleotides for the isoform without exon 2 or 3. The linear range for       To test whether antisense oligonucleotides could reverse the
PCR conditions was tested in pilot experiments with wild type and 1
                                                                             inclusion of the mutant exon 10, COS-1 cells were co-trans-
tau minigenes in the range of 20 – 40 cycles and with endogenous tau from
PC12 cells in the range of 25– 45 cycles, and bands were visualized by       fected with exon 10 minigene containing the 1 mutation and
ethidium bromide staining. The linear ranges were 28 –38 cycles for tau      increasing concentrations of antisense oligonucleotide E10 or
minigenes and 32– 42 cycles for rat tau. All subsequent reactions were       E10 , targeted to the 5 or 3 splice junction of exon 10, respec-
carried out within the linear range. Samples were separated on 2% aga-       tively (Fig. 1c). Both oligonucleotides prevented inclusion of
rose gels containing 0.5‰ ethidium bromide. Data were recorded using a       exon 10 efficiently (Fig. 1, d and f). A half-maximal effect was
Gelprint 2000i CCD imaging system and quantitated with NIH Image.
                                                                             achieved at 5–10 nM for either oligonucleotide (Fig. 1, d and f).
   Western Blotting—Proteins were extracted essentially as described
by Janke et al. (25). PC-12 cells (1.2 106 per sample) were used, and        A previously observed PCR band representing 10 /10 hetero-
the homogenization step was omitted. Dephosphorylation was per-              duplex of roughly constant intensity running below the 10
formed for 4 h at 67 °C using 18 units/ml alkaline phosphatase (Roche        band was observed in all samples and was not included in the
Molecular Biochemicals). Equal amounts of protein were separated on          quantitation (21). The exclusion of exon 10 was oligonucleotide
an 8% denaturing polyacrylamide gels and blotted onto 0.45- m nitro-         sequence-specific, since oligonucleotides containing the same
cellulose (Bio-Rad) with a Bio-Rad Mini-PROTEAN II electrophoresis
                                                                             nucleotide composition but in random order had no effect on
and Mini Trans-Blot electrophoretic transfer cell. Membranes were
probed overnight at 4 °C with anti-tau-1 (Roche Molecular Biochemi-          exon 10 inclusion at concentrations up to 200 nM (Fig. 1, e and
cals; 1:500 in PBS, 2% milk, 0.1% Tween) followed by goat anti-mouse         g). The fact that the RNA levels in control cells not treated with
horseradish peroxidase (Amersham Pharmacia Biotech; 1:1000 in PBS,           oligonucleotide and cells treated with concentrations of up to
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Correction of Tau Splicing
42988
                                                        Correction of Tau Splicing                                                           42989
200 nM oligonucleotide were identical confirms that the oligo-
nucleotides did not act through an RNA degradation mecha-
nism (Fig. 1, d and e).
   E10 contains a single mismatch to 1 tau at position 7. To
assess whether this mismatch reduced the effectiveness of the
oligonucleotide, we tested E10 on tau minigenes containing
point mutation in position 3, which, similar to the 1 muta-
tion, is also located toward the center of E10 or, alternatively,
a tau minigene with a point mutation in position 14, which is
just outside of the E10 target region (21). E10 was equally
efficient in preventing the inclusion of exon 10 into either one
of these mutated tau minigenes (Fig. 1h). We conclude that a
single mismatch in E10 to 1 tau does not significantly affect
its inhibitory efficiency.
   To ensure that this effect was specific to the tau minigene
and was not due to changes in the global splicing pattern in
COS-1 cells caused by the presence of the oligonucleotides, the
effect of E10 and E10 on the alternative splicing pattern of a
minigene containing the adenovirus 2 E1A transcript was an-
alyzed (Fig. 2). E1A contains a single 3 acceptor splice site but
multiple upstream donor sites, which give raise to 9, 10, 12,
and 13 S mRNAs (Fig. 2a) (27, 28). When COS-1 cells were
co-transfected with E1A and either one of the antisense oligo-




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nucleotides, neither oligonucleotide had any effect on E1A
splicing products (Fig. 2, b and c). Furthermore, neither oligo-
nucleotide had any effect on a minigene containing the alter-
natively spliced fibronectin-EDI exon (29) (data not shown).
Taken together, the results demonstrate that antisense oligo-
nucleotides complementary to tau exon 10 splice junctions ef-
fectively prevent inclusion of exon 10 during the splicing reac-
tion, that this effects occurs in a oligonucleotide sequence-
specific manner, and that the effect on tau exon 10 splicing is
gene-specific.
   Exclusion of Tau Exon 10 from the Endogenous mRNA—We
next sought to test whether antisense oligonucleotides can also
force exclusion of tau exon 10 in the context of the endogenous
transcript. To this end, we used rat neuronal pheochromocy-
toma PC12 cells. PC12 cells express predominantly the 10
isoform and were thus useful to test whether modified oligonu-
cleotides could shift the predominant form to the 10 isoform                   FIG. 2. Antisense oligonucleotides against tau have no effect
(30) (Fig. 3). The predominant inclusion of exon 10 in rat tau is           on alternative splicing of adenovirus E1A. a, schematic represen-
                                                                            tation of the E1A splicing reporter minigene containing multiple donor
most likely due to three nucleotide changes near the 5 splice               sites and a common acceptor site. The observed splicing isoforms are
site, which resemble stem-loop-disrupting mutations in human                indicated as 9, 10, 12, and 13 S. b and c, titration of antisense oligonu-
tau (21). PC12 cells were transfected with increasing amounts               cleotides E10 or E10 into COS-1 cells co-transfected with the E1A
of E10 or rat E10 . RatE10 is identical to E10 , except that                minigene construct 24 h after transfection. The oligonucleotides have
                                                                            no effect on E1A splicing. An additional band representing a PCR
it contains three nucleotide changes to make it fully comple-               artifact commonly seen with E1A is indicated by an asterisk. c, quan-
mentary to the rat sequence. The alternative splicing pattern of            titation of the relative amounts of E1A isoforms. Values are averages
rat tau mRNA was analyzed 24 h after transfection by RT-PCR                 from three experiments.
using primers in exons 9 and 11 (Fig. 3a). As in the minigene
system, both antisense oligonucleotides effectively excluded                Compared with the minigene system, the required concentra-
exon 10 from the tau mRNA (Fig. 3, b and d). A half-maximal                 tions were significantly higher due to lower transfection effi-
effect was achieved at 1.5 M for both E10 and rat E10 .                     ciency of PC12 cells with oligonucleotides (data not shown).



   FIG. 1. Correction of tau exon 10 inclusion in a minigene system. a, schematic representation of the tau exon 10 minigene system
consisting of the full-length human tau exon 10 (93 bp) flanked by human immunodeficiency virus tat exons. The upstream intron consists of the
311-bp tat intron (thin line) and 40-bp tau intron sequence (thick line). The downstream intron consists of 62 bp of tau intron (thick line) including
the stem-loop at the end of tau exon 10 followed by 2039 bp of tat intron (thin line). The positions of the PCR primers SD2 and SA4 within human
immunodeficiency virus tat allowing specific amplification of tau RNA from the minigene are indicated by arrows. The position of the antisense
oligonucleotides E10 and E10 are indicated by black bars. b, RT-PCR analysis of the splicing pattern of the minigene construct containing either
wild type or mutant tau exon 10 containing a U 3 C change at position 1 at the 3 end of tau exon 10. Exon 10 inclusion results in a fragment
of 246 bp; exon 10 skipping results in a fragment of 153 bp. An additional band representing a heteroduplex of 10 and 10 is indicated by an
asterisk. This band was constant between samples and was not included in the quantitation. c, sequence and location of antisense oligonucleotides
within the tau gene. Exon 10 is in capital letters, and introns are shown in lowercase letters. The position of the stem-loop is indicated by a line.
d and e, titration of antisense oligonucleotides in COS-1 cells co-transfected with the tau-1 minigene construct; d and f, oligonucleotides
complementary to the exon 10 splice junctions; e and g, a scrambled control oligonucleotide containing the same nucleotide composition but in
random order. Alternative splicing patterns were analyzed by RT-PCR 24 h after transfection. f and g, quantitation of titrations. h, titration of
E10 in COS-1 cells co-transfected with either a tau 3 or 14 minigene construct. All values represent the fraction of tau 10 isoform from at
least three experiments S.D.
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   FIG. 3. Correction of exon 10 inclusion in the endogenous tau gene in PC12 cells. a, schematic representation of the tau exon 10 region.
The positions of the PCR primers are indicated by arrows. The position of the antisense oligonucleotides E10 and rE10 are indicated by black
bars. Exon 10 inclusion results in a 367-bp fragment, and exon 10 exclusion results in a 297-bp fragment. b and c, titration of antisense
oligonucleotides into PC12 cells 24 h after transfection with antisense oligonucleotides either complementary to the exon 10 splice junctions (b and
d) or scrambled control oligonucleotides containing the same nucleotide composition but in random order (c and e). An additional band representing
a 10 /10 heteroduplex is indicated by an asterisk. Alternative splicing patterns were analyzed by RT-PCR 24 h after transfection. d and e,
quantitation of titration. Values represent the fraction of tau 10 isoform from at least three experiments S.D.


Control antisense oligonucleotides of the same base composi-               located in exons 1 and 4 (Fig. 4a). Neither E10 nor rat E10
tion as E10 or rat E10 , but in random order, had no effect on             had any effect on the splicing pattern of exons 2 and 3 in
tau splicing at concentrations up to 10 M (Fig. 3, c and e). E10           endogenous tau (Fig. 4, b and c). Similarly, neither oligonucleo-
targeted against the human tau gene had a 2–3-fold lower                   tide had any effect on alternative splicing of the ARP3 gene,
inhibitory activity than rE10 (data not shown).                            encoding an actin-related protein (31) (data not shown). These
   To test whether the effect of the antisense oligonucleotides            results show that inclusion of tau exon 10 in the context of its
was specific to tau exon 10, we probed the splicing pattern of             endogenous pre-mRNA is effectively prevented by the presence
the alternatively spliced exons 2 and 3 in tau using primers               of antisense oligonucleotides, that this effects occurs in an
                                                  Correction of Tau Splicing                                                  42991




   FIG. 4. Antisense oligonucleotides
against tau exon 10 have no effect on
alternative splicing of tau exons 2
and 3. a, schematic representation of ex-
ons 1– 4 of rat tau. Splicing isoforms ei-
ther contain exon 2 and exon 3, exon 2
alone, or neither exon 2 nor exon 3. Prim-
ers used for PCR are indicated by arrows.
b, titration of antisense oligonucleotides
E10 or rE10 in PC12 cells 24 h after
transfection. The oligonucleotides have no




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effect on exon 2 and exon 3 splicing. c,
quantitation of the relative amounts of
tau isoforms. Values are averages from
three experiments.




oligonucleotide sequence-specific manner, and that the effect       that treatment with antisense oligonucleotides increased the
on tau exon 10 splicing is specific and has no effect on other      relative amount of 3R protein in PC12 cells.
alternative splicing events in the same gene.                          Effect of Reduced 4R Tau Levels on the Cytoskeleton—Tau is
   Reduction of 4R Tau Protein—To probe whether the effect of       a microtubule-associated protein, and functional differences
antisense oligonucleotides was restricted to the RNA or was         between the 3R and the 4R isoform have been reported, al-
translated into a change in the relative levels of 3R and 4R        though the molecular basis for these differences is not entirely
protein isoforms, we performed Western blotting. PC12 cells         clear (11, 32, 33). Due to the functional differences between the
were transfected with either a control oligonucleotide of scram-    3R and the 4R isoform, altering the ratio between the two
bled sequence or either E10 or rat E10 . Cells were grown for       isoforms might affect cytoskeletal architecture. We were not
48 h and tau proteins isolated using several precipitation steps    able to reliably analyze microtubule morphology in PC12 cells
and dephosphorylated as previously described (25). All six tau      due to their small size and spherical shape. As an alternative,
protein isoforms were detected using the phosphorylation-in-        we analyzed the cytoskeletal morphology in rat AR42J pancre-
dependent anti-tau-1 antibody. As expected, three pairs of          atic acinar cells, which have previously been demonstrated to
bands were detected in PC12 cells (Fig. 5a). In each pair, the      strongly express 4R tau isoforms (34). AR42J cells were either
upper band represents the 4R isoform, and the lower band            mock-transfected or transfected with a control oligonucleotide
represents the 3R isoform (4, 25). The three pairs correspond to    of scrambled sequence or either E10 or rat E10 , and the cells
the isoforms containing exons 2 and 3 together, exon 2 only, or     were processed for microscopy (Fig. 6). As expected, in mock-
neither exon 2 nor exon 3. Exon 3 alone is never included (4). In   transfected cells or cells treated with the scrambled control
cells treated with control oligonucleotides, the 4R isoform was     oligonucleotide, predominantly the 10 tau isoform was ex-
predominant in each pair (Fig. 5a). In contrast, treatment of       pressed (Fig. 6a). In contrast, either one of the oligonucleotides
PC12 cells with E10 or rat E10 resulted in the increased            targeted to the splice junction strongly promoted exon 10 ex-
accumulation of the 3R isoform in all pairs as indicated by the     clusion and led to the predominant formation of the 10 tau
appearance of the lower bands (Fig. 5a). Quantitative analysis      isoform (Fig. 6a). By phase-contrast microscopy, cells treated
of Western blots demonstrated that the relative decrease in 4R      with control oligonucleotide were normal in appearance and
isoform varied from about 3-fold for the 2 3 isoform to about       were indistinguishable from mock-transfected control cells,
15-fold for the 2 3 isoform (Fig. 5b). These results suggest        demonstrating that the administration of oligonucleotide alone
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                                                                                 FIG. 6. Antisense oligonucleotides cause alteration in the mi-
                                                                              crotubule cytoskeleton. Rat AR42J cells were either mock-trans-
   FIG. 5. Antisense oligonucleotide treatment reduced the                    fected or transfected with 1 M of either a scrambled control oligonu-
amount of 4R protein in PC12 cells. a, PC12 cells were transfected            cleotide, E10 , or rE10 and grown for 48 h, and alternative splicing of
with antisense oligonucleotide E10 or rE10 and grown for 48 h. Tau            tau exon 10 was analyzed by RT-PCR (a) or processed for microscopy
protein was extracted and detected by Western blotting using a phos-          (b– d). a, mock-transfected AR42J cells predominantly express the exon
phorylation-independent tau antibody that recognizes all tau isoforms.        10 tau isoform. In cells treated with scrambled control oligonucleo-
Alternative inclusion of exons 2, 3, and 10 results in six protein isoforms   tide, tau exon 10 was predominantly included. Treatment of cells with
as indicated. b, quantitation of tau 4R/3R protein ratios. Treatment of       E10 or E10 prevented the inclusion of exon 10, and the 10 form of
PC12 cells with antisense oligonucleotides caused a significant reduc-        tau was predominant. b, in phase-contrast microscopy, cells treated
tion of the 4R/3R protein ratio. Values are averages from at least three      with either E10 or rE10 appeared less spread out compared with cells
experiments.                                                                  treated with a scrambled control oligonucleotide or mock-transfected
                                                                              cells. c and d, indirect immunofluorescence microscopy using an anti-
                                                                              tubulin antibody showed that in mock-transfected cells and in cells
has no effect on cell morphology (Fig. 6b). The cells showed
                                                                              treated with scrambled control oligonucleotides, the microtubule cy-
typical fibroblast-like cytoplasmic extensions, and in some cells             toskeleton was intact, and normal microtubule bundles were observed.
characteristic leading edges could be observed (Fig. 6b). In                  In contrast, cells treated with E10 or rE10 lacked microtubule cables,
contrast, cells treated with either E10 or rat E10 were round                 and tubulin appeared accumulated at the cell periphery. b, phase-
in shape, although they were still attached to the substratum                 contrast microscopy (bar, 25 m); c, fluorescence microscopy, low mag-
                                                                              nification (bar, 10 m); d, fluorescence microscopy, high magnification
(Fig. 6b). To directly visualize the microtubule cytoskeleton,                (bar, 2.3 m).
cells were stained with an anti-tubulin antibody. In mock-
transfected cells and in cells treated with scrambled control
oligonucleotide, a typical microtubule stain was observed. Mi-                inclusion of the exon. This strategy mimics the regulation of
crotubule cables of several micrometers in length were seen                   exon 10 splicing in vivo. In human brain, excessive inclusion of
throughout the whole cell, and parallel bundles were detected                 exon 10 is prevented by a stem-loop at the 3 -end of exon 10
at the periphery of cells (Fig. 6, c and d). Note that the images             formed by the terminal two bases of exon 10 and 16 bases in the
shown are confocal sections through equatorial planes of the                  adjacent intron (4, 19 –23). The stem-loop generates an unfavor-
cell and thus do not show the cables above and below the nucleus.             able binding site for the U1 small nuclear RNA and thus reduces
In contrast, in cells treated with oligonucleotides E10 or rat                the splicing efficiency of exon 10 (4, 19, 24). Loss of negative
E10 , no distinct microtubule cables were observed (Fig. 6, c and             splicing regulation by the stem-loop is critical for the disease
d). Tubulin staining was confined to the very periphery of the                process. Patient mutations in the 3 -end of human tau exon 10
cell, and no peripheral bundles could be detected. More than 90%              and the adjacent intron region contribute in two ways to exon 10
of the cell population treated with oligonucleotide exhibited this            inclusion. First, mutations destabilize the stem-loop, resulting in
phenotype. These results show that a reduction in 4R tau protein              increased access of the splicing machinery to the splice junction.
has a significant physiological effect by destabilizing the micro-            Second, all reported mutations also increase the complementar-
tubule cytoskeleton and altering cell morphology.                             ity of the 5 splice site sequence to the 5 -end of the U1 small
                                                                              nuclear RNA. The introduced antisense oligonucleotides there-
                              DISCUSSION                                      fore mimic the function of the stem-loop by impeding the access of
   We show here that modified antisense oligonucleotides com-                 U1 small nuclear ribonucleoprotein to the 3 -end of exon 10.
plementary to the 5 - and 3 -splice sites of exon 10 of tau                      Blocking of splice sites appears to be an efficient way to
pre-mRNA effectively prevent inclusion of this exon into the                  modulate splice site choice in vivo. Antisense oligonucleotides
mature mRNA during the splicing reaction. The effect is gene-                 have been used in several other systems including -globin,
specific, extends to the protein level, and induces a physiological           cystic fibrosis transmembrane conductance receptor, bcl-X, and
effect manifested by a change in the microtubule cytoskeleton.                dystrophin to block access of the splicing machinery to an
   The rationale of our approach was to block access of the                   undesirable splice site (35– 41). Whereas all reported cases of
splicing machinery to either splice junction, thus preventing                 antisense-mediated correction of splicing defects used oligonu-
                                                                  Correction of Tau Splicing                                                                          42993
cleotides targeted directly to the incorrectly used splice site, it                       3. Cooper, T. A., and Mattox, W. (1997) Am. J. Hum. Genet. 61, 259 –266
                                                                                          4. Spillantini, M. G., and Goedert, M. (1998) Trends Neurosci. 21, 428 – 433
should similarly be possible to target splicing enhancers as a                            5. Foster, N. L., Wilhelmsen, K., Sima, A. A., Jones, M. Z., D’Amato, C. J., and
mechanism to prevent inclusion of an exon. The efficiency of                                     Gilman, S. (1997) Ann. Neurol. 41, 706 –715
                                                                                          6. Goedert, M., Spillantini, M. G., and Davies, S. W. (1998) Curr. Opin. Neuro-
this approach might, however, be lower. In our hands, an                                         biol. 8, 619 – 632
antisense oligonucleotide targeted against an internal region of                          7. Wilhelmsen, K. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7120 –7121
exon 10 had a partial effect in the context of the tau minigene                           8. Hong, M., Zhukareva, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H.,
                                                                                                 Bird, T. D., McKeel, D., Goate, A., Morris, J. C., Wilhelmsen, K. C.,
but showed only a weak effect on the endogenous tau RNA.2                                        Schellenberg, G. D., Tojanowski, J. Q., and Lee, V. M.-Y. (1998) Science 282,
Similarly, it should be possible to use oligonucleotides against                                 1914 –1917
                                                                                          9. Hutton, M., Lendon, C. L., Rizzu, P., Baker, M., Froelich, S., Houlden, H.,
splicing silencers to promote inclusion of a particular exon. The                                Pickering-Brown, S., and Chakraverty, S. (1998) Nature 393, 702–705
recent development of computational methods to identify en-                              10. Clark, L. N., Poorkaj, P., Wszolek, Z., Geschwind, D. H., Nasreddine, Z. S.,
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on cell physiology by altering the microtubule cytoskeleton.                             11. Barghorn, S., Zheng-FIschhofer, Q., Ackmann, M., Biernat, J., von Bergen, M.,
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ent but rather creates a protein with distinct properties (13).                          22. D’Souza, I., Poorkaj, P., Hong, M., Nochlin, D., Lee, V. M., Bird, T. D., and
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