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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 Downloaded from www.jbc.org by guest, on March 5, 2010 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: firstname.lastname@example.com. 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- Downloaded from www.jbc.org by guest, on March 5, 2010 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 Downloaded from www.jbc.org by guest, on March 5, 2010 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- Downloaded from www.jbc.org by guest, on March 5, 2010 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. 42990 Correction of Tau Splicing Downloaded from www.jbc.org by guest, on March 5, 2010 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 Downloaded from www.jbc.org by guest, on March 5, 2010 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 42992 Correction of Tau Splicing Downloaded from www.jbc.org by guest, on March 5, 2010 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., hancers and silencers should greatly add in the selection of Miller, B., Li, D., Payami, H., Awert, F., Markopoulou, K., Andreadis, A., D'Souza, I., Lee, V. M., Reed, L., Trojanowski, J. Q., Zhurakeva, V., Bird, T., putative targets for such approaches (42, 43). Schellenberg, G., and Wilhelmsen, K. C. (1998) Proc. Natl. Acad. Sci. Modulation of the 3R/4R protein ratio had a significant effect U. S. A. 95, 13103–13107 on cell physiology by altering the microtubule cytoskeleton. 11. Barghorn, S., Zheng-FIschhofer, Q., Ackmann, M., Biernat, J., von Bergen, M., Mandelkow, E. M., and Mandelkow, E. (2000) Biochemistry 39, 11714 –11721 Upon increase of the 3R/4R ratio, microtubule cables were lost, 12. Kanai, Y., Chen, J., and Hirokawa, N. (1992) EMBO J. 11, 3953–3961 and cells appeared more spherical in shape. These observation 13. Goode, B. L., Chau, M., Denis, P. E., and Feinstein, S. C. (2000) J. Biol. Chem. 275, 38182–38189 suggests that, at least in pancreatic acinar cells, the 4R isoform 14. Buee, L., Bussiere, T., Buee-Scherrer, V., Delacourte, A., and Hof, P. R. (2000) serves to stabilize microtubules. The loss of microtubule integ- Brain Res. 33, 95–130 rity upon lowering the 4R/3R protein ratio indicates that the 15. Friedhoff, P., von Bergen, M., Mandelkow, E. M., and Mandelkow, E. (2000) Biochim. Biophys. Acta 1501, 122–132 precise balance of 4R/3R isoforms is crucial for maintenance of 16. Lee, G., Neve, R. L., and Kosik, K. S. (1989) Neuron 2, 1615–1624 an intact microtubule skeleton. This is consistent with dual 17. Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Neuron 3, 519 –526 color microscopy experiments, which have recently demon- 18. Goedert, M., Crowther, R. A., and Spillantini, M. G. (1998) Neuron 21, 955–958 strated that the two tau isoforms compete for the same binding Downloaded from www.jbc.org by guest, on March 5, 2010 19. Varani, L., Hasegawa, M., Spillantini, M. G., Smith, M. J., Murrel, J. R., sites on microtubules and that 4R isoforms can displace 3R Ghetti, B., Klug, A., Goedert, M., and Varani, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8229 – 8234 isoforms from microtubules (44). Furthermore, it is becoming 20. Hasegawa, M., Smith, M. J., Iijima, M., Tabira, T., and Goedert, M. (1999) clear that the presence of an additional microtubule-binding FEBS Lett. 443, 93–96 21. Grover, A., Houlden, H., Baker, M., Adamson, J., Lewis, J., Prihar, G., domain in the 4R isoforms does not simply have an additive Pickering-Brown, S., Duff, K., and Hutton, M. (1999) J. Biol. Chem. 274, effect to the three microtubule-binding domains already pres- 15134 –15143 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 Schellenberg, G. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5598 –5603 We suggest that the use of antisense oligonucleotides will be a 23. Gao, Q.-S., Memmott, J., Lafyatis, R., Stamm, S., Screaton, G., and Andreadis, useful tool for the study of the in vivo roles of tau isoforms. A. (2000) J. Neurochem. 74, 490 –500 24. D'Souza, I., and Schellenberg, G. D. (2000) J. Biol. Chem. 275, 17700 –17709 Modulation of the relative ratios of tau isoforms using anti- 25. Janke, C., Beck, M., Holzer, M., Bigl, V., and Arendt, T. (2000) Brain Res. 5, sense oligonucleotides will be useful to understand the role of 231–242 tau both in normal cell differentiation and in the disease proc- 26. Lamond, A. I., Sproat, B., Ryder, U., and Hamm, J. (1989) Cell 58, 383–390 ´ 27. Caceres, J. F., Stamm, S., Helfman, D. M., and Krainer, A. R. (1994) Science ess and also holds potential for therapeutic intervention in 265, 1706 –1709 FTDP-17. Mutation of the tau gene resulting in increased in- 28. Bourgeois, C. F., Popielarz, M., Hildwein, G., and Stevenin, J. (1999) Mol. Cell. Biol. 19, 7347–7356 clusion of exon 10 is now firmly established as a cause for 29. Kornblihtt, A. R., Pesce, C. G., Alonso, C. R., Cramer, P., Srebrow, A., Werbajh, FTDP-17, although not all FTDP-17 mutations result in splic- S., and Muro, A. F. (1996) FASEB J. 10, 248 –257 ing defects (4, 8, 9, 18, 24). Tau aggregates are probably a 30. Smith, C. J., Anderton, B. H., Davis, D. R., and Gallo, J. M. (1995) FEBS Lett. 375, 243–248 crucial factor in disease progression, since they induce neuro- 31. Jay, P., Berge-Lefranc, J. L., Massacrier, A., Roessler, E., Wallis, D., Muenke, nal death. Reduction of tau aggregates by increasing the 3R/4R M., Gastaldi, M., Taviaux, S., Cau, P., and Berta, P. (2000) Eur. J. Biochem. tau ratio might contribute to slowing of the disease progres- 267, 2921–2928 32. Goedert, M., and Jakes, R. (1990) EMBO J. 9, 4225– 4230 sion. Our observation that the microtubule cytoskeleton re- 33. Goode, B. L., and Feinstein, S. C. (1994) J. Cell Biol. 124, 769 –782 sponds to the increased presence of 3R tau is encouraging in 34. Vanier, M.-T., Neuville, P., MIchalik, L., and Launay, J.-F. (1998) J. Cell Sci. 111, 1419 –1432 this regard. Correction of tau splicing might also be useful as a 35. Sierakowska, H., Sambade, M. J., Agrawal, S., and Kole, R. (1996) Proc. Natl. therapeutic approach to other neurological disorders. Patholog- Acad. Sci. U. S. A. 93, 12840 –12844 ical tau proteins similar to the ones found in FTDP-17 are also 36. Lacerra, G., Sierakowska, H., Carestia, C., Fucharoen, S., Summerton, J., Weller, D., and Kole, R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9591–9596 present in corticobasal degeneration and in progressive su- 37. Dunckley, M. G., Manoharan, M., Villiet, P., Eperon, I. C., and Dickson, G. pranuclear palsy (4, 6, 45– 47). A role for alternative splicing (1998) Hum. Mol. Genet. 7, 1083–1090 38. Mann, C. J., Honeyman, K., Cheng, A. J., Ly, T., Lloyd, F., Fletcher, S., defects in these disease has not been demonstrated but can be Morgan, J. E., Partridge, T. A., and Wilton, S. D. (2001) Proc. Natl. Acad. suspected based on the finding that a genetic polymorphism in Sci. U. S. A. 98, 42– 47 the intron preceding tau exon 10 has been reported in progressive 39. Wilton, S. D., Lloyd, F., Carville, K., Fletcher, S., Honeyman, K., Agrawal, S., and Kole, R. (1999) Neuromusc. Disorders 9, 330 –338 supranuclear palsy (48, 49). Despite the unsolved challenge of 40. Friedman, K. J., Kole, J., Cohn, J. A., Knowles, M. R., Silverman, L. M., and delivery of oligonucleotides to the brain, our results suggest that Kole, R. (1999) J. Biol. Chem. 274, 36193–36199 41. Taylor, J. K., Zhang, Q. Q., Wyatt, J. R., and Dean, N. M. (1999) Nature antisense-based strategies might be used in therapeutic ap- Biotechnol. 17, 1097–1100 proaches for neurological disorders involving tau aggregates. 42. Liu, H. X., Zhang, M., and Krainer, A. R. (1998) Genes Dev. 12, 1998 –2012 43. Liu, H. X., Cartegni, L., Zhang, M. Q., and Krainer, A. R. (2001) Nat. Genet. 27, Acknowledgments—We thank A. Grover, M. Hutton, J. Caceres, A. 55–58 Kornblihtt, P. Jay, and S. Agrawal (Hybridon) for reagents and C. 44. Lu, M., and Kosik, K. S. (2001) Mol. Biol. Cell 12, 171–184 45. Ksiezak-Reding, H., Morgan, K., Mattiace, L. A., Davies, P., Liu, W. K., Yen, Baker, R. Kole, and J. Caceres for technical advice and critical com- S. H., Weidenheim, K., and Dickson, D. W. (1994) 145, 1496 –1508 ments on the manuscript. 46. Flament, S., Delacourte, A., Verny, M., Hauw, J. J., and Javoy-Agid, F. (1991) Acta Neuropathol. 81, 591–596 REFERENCES 47. Ishizawa, K., Ksiezak-Reding, H., Davies, P., Delacourte, A., Tiseo, P., Yen, 1. Hanke, J., Brett, D., Zastrow, I., Aydin, A., Delbruck, S., Lehmann, G., Luft, F., S. H., and Dickson, D. W. (2000) Acta Neuropathol. 100, 235–244 Reich, J., and Bork, P. (1999) Trends Genet. 15, 389 –390 48. Conrad, C., Andreadis, A., Trojanowski, J. Q., Dickson, D. W., Kang, D., Chen, 2. Philips, A. V., and Cooper, T. A. (2000) Cell. Mol. Life Sci. 57, 235–249 X., Wiederholt, W., Hansen, L., Masliah, E., Thal, L. J., Katzman, R., Xia, Y., and Saitoh, T. (1997) Ann. Neurol. 41, 277–281 49. Higgins, J. J., Litvan, I., Pho, L. T., Li, W., and Nee, L. E. (1998) Neurology 50, 2 B. Kalbfuss and T. Misteli, unpublished result. 270 –273
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- Characterisation of signal transduction pathways in proliferating vascular smooth muscle cells and their correlation with the process of arteriogenesis (2003)
- Tomato heat stress transcription factor HsfA1 as master regulator of thermotolerance (2003)
- The heat stress (hs) response is universal to all organisms. As the cell senses increase in temperature, heat stress transcription factors (Hsfs) are activated to upregulate the expression of a number of genes encoding heat stress proteins (Hsp) which act as molecular chaperones to protect cells against heat damages. In higher plants, the phenomenon seems to be unusually complex both at the level of Hsfs and Hsps (e.g., 21 Hsf encoding genes in Arabidopsis and at least 17 in tomato). Upon prolonged hs, another characteristic property of plant cells is the assembly of large cytosolic aggregates called heat stress granules (HSG), which are composed of Hsps, HsfA2, RNA and RNA-binding proteins. The present work was aimed to understand plant hs response using tomato as a model system. To study the function of tomato Hsfs in their native system, we generated transgenic tomato lines altered in expression of HsfA1, HsfA2, and HsfB1. Tomato plants with 10-fold overexpression of HsfA1 (OE plants) were characterised by integration of a single HsfA1 expression cassette, whereas the plants harbouring a tandem inverted repeat (IR) of the cassette showed cosuppression of HsfA1 (CS plants). The lack of HsfA1 expression in CS plants results from posttranscriptional gene silencing connected with the formation of small interfering RNA (siRNA). Under normal growth conditions, major developmental features were similar for wild-type (WT), OE and CS plants. However, in contrast to the former two, CS plants and fruits were extremely sensitive to elevated temperature because hs-induced synthesis of major chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family, the function of tomato HsfA1 is unique as master regulator of induced thermotolerance. On the other hand, maintenance of essential chaperones in CS plants during seed development suggests involvement of other Hsfs and/or transcription factor(s). HsfB1 and HsfA2 transgenic tomato plants, unaffected in thermotolerance, further supported the function of HsfA1 as the major factor regulating hs-inducible genes. Hs87 independent phenotypes of plants with altered expression of HsfB1 indicates developmental role of this Hsf. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmids encoding Hsfs A1, A2 and A3 were well expressed which could function as activators for reporter gene expression. However, in protoplasts derived from CS plants, plasmids encoding HsfA2 and HsfA3 were normally expressed but even higher amounts of HsfA1 expression plasmids were completely silenced. Therefore, silencing of HsfA1 in CS plants was also reproduced in its mesophyll protoplasts. Lacking thermotolerance in CS protoplasts could be restored after transformation with expression plasmids encoding functionally equivalent HsfA2 or HsfA3 resulting in (i) expression of chaperones, (ii) survival of the cells at otherwise lethal temperature, (iii) thermoprotection of firefly luciferase, and (iv) assembly of heat stress granules (HSGs). The strong silencing caused by an IR in CS plants opened the possibility of a broad use of RNAi for gene knock-down also in the transient system of mesophyll protoplasts. Using this technology, we attempted to dissect essential components of thermotolerance and HSG assembly. We demonstrated the previously reported function of chaperones such as Hsp70 and Hsp101, and could discriminate the in vivo chaperone functions of different isoforms of Hsp20 and Hsp70 proteins. Hsp17-CI, Hsp70 (hs-inducible isoforms), and Hsp101 are absolutely essential chaperones for thermotolerance in plants. Furthermore, the results also show that despite Hsp17-CI and -CII being major components of HSG complexes, they are dispensable for assembly of these complexes. Based on these results, it is proposed that in the transient protoplast system an approach with gene-specific IRs can be used to discriminate functions of closely related isoforms among protein-families and to dissect complex protein networks.


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Posted at: 03/16/2013 6:52 PM
SIOUX CITY, Iowa (AP) - A Sioux City gynecologist has been fined $7,500 for botching a hysterectomy by mistakenly removing his 29-year-old patient's healthy ovaries.
The Sioux City Journal reports (http://bit.ly/ZQ6gZ6) that the Iowa Board of Medicine fined 50-year-old Dr. Kevin Hamburger on Thursday for the July 2011 surgery and has ordered Hamburger to submit a plan describing how he'll avoid future errors.
Hamburger is also being sued for damages by the patient, Erin Rehan. Her lawsuit says she was told by Hamburger that her ovaries would not be removed during the procedure.
Rehan is seeking more than $10,000 in damages.