Cyclic AMP-dependent Protein Kinase Regulates the Alternative Splicing of Tau Exon 10: A MECHANISM INVOLVED IN TAU PATHOLOGYOFALZHEIMER DISEASE

Cyclic AMP-dependent Protein Kinase Regulates theAlternative Splicing of Tau Exon 10A MECHANISM INVOLVED IN TAU PATHOLOGY OF ALZHEIMER DISEASE*□S

Received for publication, November 19, 2010, and in revised form, February 15, 2011 Published, JBC Papers in Press, March 2, 2011, DOI 10.1074/jbc.M110.204453

Jianhua Shi‡§, Wei Qian‡§, Xiaomin Yin‡§, Khalid Iqbal¶, Inge Grundke-Iqbal¶, Xiaosong Gu‡, Fei Ding‡,Cheng-Xin Gong¶, and Fei Liu‡¶1

From the ‡Jiangsu Key Laboratory of Neuroregeneration and §Department of Biochemistry, Medical School, Nantong University,Nantong, Jiangsu 226001, China and the ¶Department of Neurochemistry, New York State Institute for Basic Research inDevelopmental Disabilities, Staten Island, New York 10314

Hyperphosphorylation and deposition of tau into neurofibril-lary tangles is a hallmark of Alzheimer disease (AD). Alternativesplicing of tau exon 10 generates tau isoforms containing threeor fourmicrotubule binding repeats (3R-tau and 4R-tau), whichare equally expressed in adult human brain. Dysregulation ofexon 10 causes neurofibrillary degeneration. Here, we reportthat cyclic AMP-dependent protein kinase, PKA, phosphory-lates splicing factor SRSF1, modulates its binding to taupre-mRNA, andpromotes tau exon10 inclusion in cultured cellsand in vivo in rat brain. PKA-C�, but not PKA-C�, interactswith SRSF1 and elevates SRSF1-mediated tau exon 10 inclusion.In AD brain, the decreased level of PKA-C� correlates with theincreased level of 3R-tau. These findings suggest that a down-regulation of PKA dysregulates the alternative splicing of tauexon 10 and contributes to neurofibrillary degeneration in ADby causing an imbalance in 3R-tau and 4R-tau expression.

Tau is a neuronal microtubule-associated protein, the func-tion of which is to stimulatemicrotubule assembly and stabilizemicrotubules. Hyperphosphorylation of tau leads to its aggre-gation into neurofibrillary tangles, a hallmark of Alzheimer dis-ease (AD)2 and related neurodegenerative diseases calledtauopathies (1–4). Adult human brain expresses six differenttau isoforms from a single gene by alternative splicing of exons2, 3, and 10 of its pre-mRNA (5). The exon 10 encodes thesecond microtubule binding repeat (6). Alternative splicing ofexon 10 generates tau with three or four microtubule bindingrepeats (3R-tau or 4R-tau), which is under developmental andcell type-specific regulation. Only 3R-tau is expressed duringembryogenesis, whereas 3R-tau and 4R-tau are expressed inapproximately equal amounts in adult human brain (6, 7). Sev-

eral mutations in tau gene result in either an increase or adecrease in 4R-tau expression and cause frontotemporaldementia with parkinsonism linked to chromosome 17 (FTDP-17), one of the tauopathies (8). Thus, alteration in the 3R-tau/4R-tau ratio is sufficient to trigger neurodegeneration in fron-totemporal dementia and might also play a role in otherneurodegenerative disorders such as Pick’s disease, progressivenuclear palsy, or corticobasal degeneration in which the3R-tau/4R-tau ratio is markedly altered (9–12). Thus, the reg-ulation of alternative splicing of human tau exon 10 has been ofcritical interest. However, results of studies of the alternativesplicing of tau exon 10 in AD brain have been contradictory(13–15). Recent studies have shown that aggregation and dep-osition of 3R-tau may be associated with more advanced stages(16, 17).Alternative splicing of tau exon 10 is regulated by several

trans-acting factors, including serine- and arginine-rich (SR)proteins, and their phosphorylation (18–24). Splicing factor2/alternative splicing factor (ASF/SF2), nownamed SRSF1 (ser-ine/arginine-rich splicing factor 1) (25), is a prototypical SRprotein that participates in both constitutive and alternativesplicing (26). SRSF1 acts on a polypurine enhancer (PPE) ofexonic splicing enhancer located at tau exon 10 and plays essen-tial and regulatory roles in the alternative splicing of tau exon 10(24). Overexpression of SRSF1 promotes exon 10 inclusion (20,24, 27). SRSF1 contains two copies of an N-terminal RNA rec-ognition motif (RRM) and a C-terminal RS domain. The serineresidues of the RS domain are targets of phosphorylation bymultiple kinases, including SRPK1 (28), SRPK2 (29), Clk/Sty(30), DNA topoisomerase I (31), AKT (32), and Dyrk1A (27).Phosphorylation of SRSF1 regulates its translocation betweenthe cytoplasm and the nucleus or within the nucleus and affectsits function (27, 28, 30, 33–36).Cyclic AMP (cAMP)-dependent protein kinase, PKA, has

emerged as a key kinase that is able to interact withmany of theproteins involved in the etiology of AD as well as other tauopa-thies. It has been shown that PKA phosphorylates tau at severalsites and primes phosphorylation of tau by glycogen synthasekinase-3� (37). PKA is a tetrameric holoenzyme consisting oftwo catalytic (C) subunits and two regulatory (R) subunits in theabsence of cAMP. Stimulation by cAMPdissociates the holoen-zyme and causes translocation to the nucleus of a fraction ofthe C subunit. Apart from regulation of transcription, little is

* This work was supported, in whole or in part, by National Institutes of HealthGrants AG027429 (to C.-X. G.) and AG019158 (to K. I.). This work was sup-ported by National Natural Science Foundation of China Grants 30973143and 81030059 (to F. L.) and 30801202 (to J. S.), Natural Science Foundationof Jiangsu Province, China, Grant BK2009159 (to F. L.), and U. S. Alzheimer’sAssociation Grant NIRG-08-91126 (to F. L.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 6.

1 To whom correspondence should be addressed. Tel.: 718-494-4820; Fax:718-494-1080; E-mail: [email protected].

2 The abbreviations used are: AD, Alzheimer disease; SRSF1, serine/arginine(SR)-rich splicing factor 1; PPE, polypurine enhancer; CREB, cAMP-respon-sive element-binding protein; RRM, RNA recognition motif.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 16, pp. 14639 –14648, April 22, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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known about the function of the C subunit in the nucleus. It isknown that PKA phosphorylates several splicing factors and isinvolved in the pre-mRNA splicing (30, 38, 39). We recentlyfound that inADbrain, the activity of PKA is down-regulated asa result of proteolysis of the regulatory subunit by over-acti-vated calpain I (40). However, the role of PKA in the alternativesplicing of tau exon 10 was unclear. In the present study wedemonstrate that PKA phosphorylates SRSF1 and therebyenhances the inclusion of tau exon 10 and that down-regulationof PKA in AD brain correlates with increase in 3R-tau expres-sion. These results suggest that PKA is involved in the taupathology in AD via regulation of tau exon 10 splicing.

EXPERIMENTAL PROCEDURES

Human Brain Tissue—Medial frontal cortices from 15 ADand 15 age-matched normal cases used for this study (Table 1)were obtained from the Sun Health Research Institute Dona-tion Program (Sun City, AZ); all cases were histopathologicallyconfirmed. The tissue was stored at �70 °C until used. The useof frozen human brain tissue was in accordance with theNational Institutes of Health guidelines and was approved byour institutional review committee.Plasmids, Antibodies, and Other Reagents—The expression

constructs for human PKA-C� and PKA-C�were generated byreverse transcription PCR from RNA isolated from normalhuman neuronal progenitor cells and confirmed by DNAsequence analysis. Both PKA-C� and PKA-C� tagged with HAwere cloned into pCI-Neo vector via SalI andNotI sites. pCEP4-SRSF1-HA was a gift from Dr. Tarn of the Institute of Biomed-ical Sciences, Academia Sinica, Taiwan. pEGFP-N1-SRSF1was generated by inserting the cDNA of SRSF1 without a stopcode into pEGFP-N1. pGEX-2T-SRSF1, pGEX-2T-SRSF1�RS2,pGEX-2T-SRSF1�RS, pGEX-2T-SRSF1RRM, and pGEX-2T-SRSF1RS were constructed by PCR amplification from pCEP4-SRSF1-HA and subcloning into pGEX-2T to express GSTfusion proteins of SRSF1 and its deletionmutants. Formamma-lian vectors, SRSF1 mutants were constructed by digestion ofthose SRSF1 mutants in pGEX-2T vector and inserting theminto pCEP4 to generate pCEP4-SRSF1 deletion mutants taggedwith HA. pCI-SI9/LI10 containing a tau minigene, SI9/LI10,comprising tau exons 9, 10, and 11 and part of intron 9 and thefull-length of intron 10, was a gift fromDr. Jianhua Zhou of theUniversity ofMassachusettsMedical School. pFC-CRE-lucifer-ase was bought from Stratagene (La Jolla, CA). The catalyticsubunit of PKA, the monoclonal anti-HA, anti-�-tubulin, andanti-�-actin were bought from Sigma. Monoclonal anti-3R-tau(RD3) and anti-4R-tau (RD4) were fromUpstate Biotechnology(Lake Placid, NY). Monoclonal anti-tau (tau-5) was fromChemicon International, Inc. (Pittsburgh, PA). Monoclonalanti-human tau (43D) and polyclonal anti-tau (R134d) weredescribed previously (41). Polyclonal Anti-PKA-C�, polyclonalanti-PKA-C�, and monoclonal anti-SRSF-1 were from SantaCruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugatedanti-mouse and anti-rabbit IgG were obtained from JacksonImmunoResearch Laboratories (West Grove, PA); Alexa 488-conjugated goat anti-mouse IgG, and Alexa 555-conjugatedgoat anti-rabbit IgG and TO-PRO-3 iodide (642/661) werefrom Invitrogen (Invitrogen). The ECL kit was from Thermo

Fisher Scientific (Rockford, IL), and [�-32P]ATP and[32P]orthophosphate were from MP Biomedicals (Irvine, CA).The dual-luciferase reporter assay system was purchased fromPromega (Madison, WI).Cell Culture and Transfection—COS-7, HEK-293T, HEK-

293FT, HeLa, and CHO cells were maintained in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10%fetal bovine serum (Invitrogen) at 37 °C. All transfections wereperformed in triplicate with FuGENE 6 (Roche Diagnostics) in12-well plates. The cells were transfected with FuGENE 6 for48 h according to the manufacturer’s instructions.In Vitro Phosphorylation of SRSF1 by PKA—GST-SRSF1,

GST-SRSF1 mutants, or as a control, GST (0.2 mg/ml) wasincubatedwith various concentrations of PKA catalytic subunitin a reaction buffer consisting of 50 mM HEPES, pH 6.8, 10 mM

�-mercaptoethanol, 10 mM MgCl2, 1.0 mM EGTA, and 0.2 mM

[�-32P]ATP (500 cpm/pmol). After incubation at 30 °C for 30min, the reaction was stopped by boiling with an equal volumeof 2� Laemmli sample buffer. The reaction products were sep-arated by SDS-PAGE. Incorporation of 32P was detected byexposure of the dried gel to phosphor-image system.Phosphorylation of SRSF1 in Cultured Cells—CHOcells were

transfected with pCEP4-SRSF1-HA and cultured in DMEMsupplemented with 10% fetal bovine serum. After 45 h of trans-fection, the medium was replaced with [32P]orthophosphate(10 mCi) in DMEM (without phosphate) supplemented with10% fetal bovine serum. After a 3-h incubation, the cells wereharvested in lysate buffer (50 mM Tris-HCl, pH 7.4, 150 mM

NaCl, 50 mM NaF, 1 mM Na3VO4, 50 mM okadaic acid, 0.1%Triton X-100, 0.1% Nonidet P-40, 0.25% sodium deoxycholate,2mMEDTA, 1mMPMSF, and 10�g/ml of aprotinin, leupeptin,and pepstatin). Insoluble materials were removed by centrifu-gation, and the supernatant was incubated with anti-HA pre-coupled to protein G-conjugated agarose for 4 h at 4 °C. Afterbeing washed with TBS (50 mM Tris-HCl, pH 7.4, 150 mM

NaCl), the immunoprecipitated SRSF1-HA by anti-HA wasanalyzed by immunoblotting and autoradiography.GST Pulldown of PKA by SRSF1—GST, GST-SRSF1, and

GST-SRSF1 deletionmutants were purified by affinity purifica-tion with glutathione-Sepharose but without elution from thebeads. These beads coupled with GST, GST-SRSF1, and GSTSRSF1 deletion mutants were incubated with crude extractfrom rat brain homogenate in buffer (50 mM Tris-HCl, pH 7.4,8.5% sucrose, 50 mM NaF, 1 mM Na3VO4, 0.1% Triton X-100, 2mM EDTA, 1 mM PMSF, 10 �g/ml aprotinin, 10 �g/ml leupep-tin, and 10 �g/ml pepstatin). After 4 h of incubation at 4 °C, thebeads were washed with washing buffer (50 mM Tris-HCl, pH7.4, 150 mMNaCl, and 1 mM DTT) 6 times, the bound proteinswere eluted by boiling in Laemmli sample buffer, and the sam-ples were subjected to Western blot analysis.Co-immunoprecipitation of PKA by SRSF1—HEK-293FT

cells were transfected with pCEP4-SRSF1-HA for 40 h asdescribed above and treated with 10 �M forskolin for 8 h, andthen the cells were washed twice with PBS and lysed by sonica-tion in lysate buffer (50 mM Tris-HCl, pH 7.4, 150 mMNaCl, 50mM NaF, 1 mM Na3VO4, 2 mM EDTA, 1 mM PMSF, 2 �g/mlaprotinin, 2 �g/ml leupeptin, and 2 �g/ml pepstatin). The celllysate was centrifuged at 16,000 � g for 10 min and incubated

PKA Regulates the Alternative Splicing of Tau Exon 10

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with anti-HA overnight at 4 °C, and then protein G beads wereadded. After 4 h of incubation at 4 °C, the beads were washedwith lysate buffer twice andwithTBS twice, and boundproteinswere eluted by boiling in Laemmli sample buffer. The sampleswere subjected toWestern blot analysis with the indicated pri-mary antibodies.Co-localization of PKA with SRSF1—HeLa cells were plated

in 24-well plates onto coverslips 1 day before transfection at50–60% confluence. The cells were then transfected withpEGFP-N1-SRSF1 as described above. After 40 h of transfec-tion, the cells were treated with 10 �M forskolin for 30 min toactivate PKA, and then the cells were washed with PBS andfixed with 4% paraformaldehyde in PBS for 30 min at roomtemperature. After washing with PBS, the cells were blockedwith 10% goat serum in 0.2% Triton X-100, PBS for 2 h at 37 °Cand incubated with mouse anti-PKA-C� (1:50) overnight at4 °C. The cells were then washed and incubated for 1 h withsecondary antibody (Alexa 488-conjugated goat anti-mouseIgG, 1:1000) plus TO-PRO-3 iodide at room temperature. Thecells were washed with PBS, mounted with Fluoromount-G,and observed with a Nikon TCS-SP2 laser-scanning confocalmicroscope.Knockdown of SRSF1 or PKA Catalytic Subunits, C� and C�,

with RNA Interference—For inhibition of SRSF1 expression,HEK-293T cells cultured in 12-well plates were transfectedwith various amounts of short interfering RNA (siRNA) usingLipofectamine 2000. After 48 h transfection, cells were lysed,and protein andRNAwere extracted as described above. siRNAis a pool of 3 target-specific 20–25-nucleotide siRNAs to knockdown target gene expression (Santa Cruz Biotechnology). Bothstrands of siRNAs had 3�-dTdT tails. The same amount ofscramble siRNA was used for controls.Quantitation of tau Exon 10 Splicing by Reverse Transcrip-

tion-PCR (RT-PCR)—Total cellular RNAwas isolated from cul-tured cells by using the RNeasy Mini Kit (Qiagen, GmbH, Ger-many). One microgram of total RNA was used for first-strandcDNA synthesis with oligo-(dT)15–18 by using the OmniscriptReverse Transcription kit (Qiagen). PCR was performed byusing PrimeSTARTM HS DNA Polymerase (Takara Bio Inc.,Otsu, Shiga, Japan) with forward primer 5�-GGTGTCCACTC-CCAGTTCAA-3� and reverse primer 5�-CCCTGGTTTA-TGATGGATGTTGCCTAATGAG-3� to measure alternativesplicing of tau exon 10. The PCR conditions were: 98 °C for 3min, 98 °C for 10 s, and 68 °C for 40 s for 30 cycles and then68 °C 10min for extension. The PCRproducts were resolved on1.5% agarose gels and quantitated using the Molecular Imagersystem (Bio-Rad).Electrophoretic Mobility Shift Assay (EMSA)—The RNA

oligonucleotides of tau cover PPE at the tau exon (5�-GUGCA-GAUAAUUAAUAAGAAGCUGGAUCUU-3�, tau-RNA) ordeleted PPE (5�-GUGCAGAUAAUUAAUGAUCUUAGCAA-CGUC-3�, tau-RNA�PPE), or deleted SC35-like element (5�-GUAUUAAUAAGAAGCUGGAUCUU-3�, tau-RNA�SC35-like)was labeled with [�-32P]ATP (3000 Ci/mM, Amersham Biosci-ences) using T4 polynucleotide kinase (New England Biolabs)and subsequently purified with MicroSpin G-25 column(Amersham Biosciences). To perform the EMSA assay, weimmunoprecipitated SRSF1-HA from HEK-293T cells trans-

fected with pCEP4-SRSF1 by using anti-HA cross-linked toprotein G-agarose with a Seize X Protein G affinity kit. Theimmunopurified SRSF1 in 50mMTris-HCl buffer, pH 7.5, con-taining 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol wasmixed with 32P-labeled tau-RNA or tau-RNA�PPE or tau-RNA�SC35-like in a total volume of 10 �l and incubated at 30 °Cfor 40 min. The mixture was separated by electrophoresis witha 6% non-denaturing polyacrylamide gel, which was pre-run at100V for 10min inTBEbuffer (89mMTris borate, pH8.0, 2mM

EDTA) at 100 V for 60 min. The gel was dried and autoradio-graphed with a PhosphorImager (Molecular Dynamics).Tau Pre-mRNA Immunoprecipitations by SRSF1—Tau RNA

immunoprecipitation was performed as described previously(42). Briefly, HEK-293FT cells were transfected with pCEP4-SRSF1-HA for 44 h followed by treatment with 10mM forskolinfor 4 h. Cells were collected in PBS and cross-linked with 1%formaldehyde for 10 min at room temperature. After quench-ing with glycine, the cells were lysed in buffer A (5 mM PIPES,pH 8.0, 85 mM KCl, 0.5% Nonidet P-40, 1� protease inhibitorsmixture, and 50 units/ml RNasin� Plus RNase Inhibitor (Pro-mega)) on ice for 10 min and centrifuged at 2000 � g for 5 minto pellet nuclei. The nuclear fraction was sonicated in buffer B(50 mM Tris-HCl, pH 8.1, 1% SDS, 10 mM EDTA, 1� proteaseinhibitorsmixture, and 50 units/ml RNasin� Plus RNase Inhib-itor). After centrifugation at 16,000 � g for 10 min, the super-natant was subject to immunoprecipitation with anti-HA in IPbuffer (16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 0.01% SDS,1.1% Triton X-100, 1.2 mM EDTA, 1� protease inhibitors mix-ture, and 50 units/ml RNasin� Plus RNase Inhibitor) for 2 h.Immune complex was washed sequentially with low salt buffer(20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, and 2mMEDTA),with high salt buffer (20mMTris-HCl,pH 8.1, 500 mM NaCl, 0.1% SDS, 1% Triton X-100, and 2 mM

EDTA), with LiCl buffer (10mMTris-HCl, pH8.1, 250mMLiCl,1%Nonidet P-40, 1% deoxycholate, and 1mM EDTA), and with10 mM Tris pH 8.0, 1 mM EDTA. Immune complex was elutedwith elution buffer (1% SDS, 0.1 M NaHCO3, and 50 units/mlRNasin� Plus RNase Inhibitor). The cross-linking was reversedby incubation with 200 mM NaCl at 65 °C for at least 2 h. Afterdigestion with 20mMTris-HCl, pH 6.5, 0.5 M EDTA, 0.8mg/mlProteinase K (Invitrogen), RNA was extracted by the RNeasyMini kit (Qiagen) and subjected to first-strand cDNA synthesiswith oligo-(dT)15–18 by using the Omniscript Reverse Tran-scription kit (Qiagen). cDNA was amplified by PrimeSTARTM

HSDNA Polymerase (Takara Bio Inc.) with two sets of primersagainst pre-mRNA of tau: primer set 1 (forward (5�-AGGC-GGGTCCAGGGTGGCGTGTCACTCATC-3�) and reverse(5�-CTAATAATTCAAGCCACAGCACGGCGCATGGGACG-3�), and primer set 2 (forward (5�-ATGCCTTCAGAGCAGC-CCTCTATCC-3�) and reverse (5�-ACGTGTGAAGGTACT-CACACTGCCG-3�)). The PCR products were resolved in 2.0%agarose gels.Intracerebroventricular Injection—Sprague-Dawley rats at

postnatal day 10 (P10) were first anesthetized by wrapping inice pack for 5 min, and then 2 �l of 1 mM forskolin in artificialCSF was injected into the left lateral ventricle of the brain ataround 2.5 mm depth. The control animals were treated iden-tically but with vehicle only. Rats were killed 3 days after injec-



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tion. The brains were immediately removed and processed formeasuring the tau exon 10 splicing by RT-PCR.Statistical Analysis—Where appropriate, the data are pre-

sented as the means � S.D. Data points were compared by theunpaired two-tailed Student’s t test, and the calculated p valuesare indicated in the figures. For analysis of the correlationbetween levels of PKA-C� and 3R-tau or 4R-tau in humanbrainhomogenates, the Pearson product-moment correlation coeffi-cient r was calculated.

RESULTS

Activation of PKA Promotes Tau Exon 10 Inclusion in HEK-293TCells and in Rat Brain—To elucidate the regulation of thealternative splicing of tau exon 10 by PKA, we transfectedmini-tau gene pCI/SI9-LI10 (comprising tau exons 9, 10, and 11 andpart of intron 9 and the full-length of intron 10) intoHEK-293Tcells and treated themwith 10 �M forskolin to activate PKA fordifferent times or with various concentrations of this activatorfor 8 h.Within 48 h of transfection, the total RNAwas extractedand subjected to RT-PCR tomeasure tau exon 10 splicing. PKAactivation with forskolin treatment activated PKA, which wasdemonstrated by phosphorylation of CREB (cAMP-responsiveelement-binding protein) and by increased luciferase activity(Fig. 1a). Interestingly, we observed that activation of PKA by

forskolin promoted tau exon 10 inclusion in a time-dependentand dose-dependent manner (Fig. 1, b and c).

Mammalian brain expresses bothC� andC� of PKA (40, 43).PKA-C� is expressed in testis only (44). To understand whichisoform of PKA regulates tau exon 10 splicing, we co-trans-fected PKA-C� or -C� with mini-tau gene pCI/SI9-LI10 intoHEK-293FT cells and analyzed the tau exon 10 splicing. Wefound that overexpression of PKA-C� significantly promotedtau exon 10 inclusion, but overexpression of PKA-C� slightlyinhibited tau exon 10 inclusion (Fig. 1d). Knock-down ofPKA-C� expression (supplemental Fig. 1) did not affect thealternative splicing at basal level but significantly inhibited for-skolin-promoted tau exon 10 inclusion (Fig. 1e). Down-regula-tion of PKA-C� by siRNA (supplemental Fig. 1) increased thetau exon 10 inclusion at basal level but did not influence forsko-lin promoted tau exon 10 inclusion (Fig. 1e). To determine thatoverexpression or knockdown of PKA catalytic subunit affectsPKAactivity, wemeasuredCREBphosphorylation at Ser-133 inthe cells transfected with PKA-C� or PKA-C� or their siRNAs.We observed that knockdown of PKA-C� or PKA-C� signifi-cantly decreased PKA activity, and overexpression of PKA-C�

or PKA-C� increased PKA activity, as determined by phosphor-ylation of CREB (supplemental Fig. 2). Taken together, these

FIGURE 1. Activation of PKA promotes tau exon 10 inclusion. a, activation of PKA by forskolin is shown. HEK-293T cells were transfected with pFC-CRE-luciferase (Stratagene) for 42 h and treated with 10 �M forskolin (Fors) for 8 h. The cell lysates were analyzed for phosphorylation of CREB at Ser-133 and totalCREB by Western blots or luciferase activity with a commercial assay system (Promega). b and c, promotion of tau exon 10 inclusion by forskolin is shown.HEK-293T cells transfected with pCI-SI9/LI10 mini-tau gene were treated with 10 �M forskolin for various times (b) or with various concentrations of forskolinfor 8 h (c). The total RNA was then extracted and subjected to RT-PCR to measure the tau exon 10 splicing. Results represent the mean � S.D. d, HEK-293FT cellswere co-transfected with pCI-SI9/LI10 mini-tau gene and PKA-C� or C� for 48 h, and the splicing products of tau exon 10 were analyzed with RT-PCR. e,HEK-293FT cells were co-transfected pCI-SI9/LI10 with siRNA of PKA-C� or PKA-C�. After 36 h of transfection, the cells were treated with 10 �M forskolin for 12 h,and the splicing of tau exon 10 was analyzed with RT-PCR. f, forskolin was injected into the left lateral ventricle of rats at postnatal day 10 (P10). Total RNA of ratbrains was extracted 3 days after injection and subjected to RT-PCR for measurement of tau exon 10 splicing. Results represent the mean � S.D.; *, p � 0.05; **,p � 0.01.



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results suggest that PKA-C� promotes andPKA-C� suppressestau exon 10 inclusion and that promotion of forskolin on tauexon 10 inclusion is dependent on PKA-C� but not PKA-C�.

Adult murine brain only expresses 4R-tau, and both 3R-tauand 4R-tau are expressed during P5 to P30 (supplemental Fig.3). To determine the correlation of developmental expressionsof PKA-C� and tau isoforms, wemeasured the level of PKA-C�in the rat brain, and we found that PKA-C� expression coin-cided with the expression of 3R-tau and 4R-tau (supplementalFig. 3). To test whether PKA regulates the alternative splicing oftau exon 10 in vivo, we injected forskolin (2 �l of 1mM) into therat left lateral ventricle at p10 and then measured the tau exon10 splicing by RT-PCR 3 days after the injection.We detected asignificant increase of 4R-tau expression in forskolin-treatedrats (Fig. 1f), which provides evidence that PKA regulates thealternative splicing of tau exon 10 in vivo and activation of PKAenhances tau exon 10 inclusion.PKA Enhances SRSF1-mediated Tau Exon 10 Inclusion—

Splicing factor SRSF1 plays a critical role in the alternativesplicing of tau exon 10. Overexpression of SRSF1 promoted tauexon 10 inclusion in a time-dependent manner (supplementalFig. 4). To determine whether PKA modulates SRSF1-pro-moted tau exon 10 inclusion, we treated SRSF1-expressingHEK-293T cells with 10�M forskolin for 8 h and thenmeasuredexon 10 splicing. We observed that treatment with forskolin aswell as SRSF1 overexpression promoted tau exon 10 inclusion,and forskolin treatment further enhanced SRSF1-promoted tauexon 10 inclusion (Fig. 2a). In contrast, knockdown of theexpression of SRSF1 (supplemental Fig. 5) by siRNA suppressedtau exon 10 inclusion and eliminated the effect of forskolin ontau exon 10 inclusion (Fig. 2b). These findings suggest that PKAmodulates SRSF1-promoted tau exon 10 inclusion.To determine the isoform-specific role of PKA catalytic sub-

units on SRSF1-mediated tau exon inclusion, we overexpressedPKA-C� and -C� in pCEP4/SRSF1-transfected HEK-293FTcells and determined tau exon 10 splicing. Consistent with thefindings shown in Fig. 1e, we found that PKA-C�, but not PKA-C�, further enhanced the SRSF1-promoted tau exon 10 inclu-sion (Fig. 2c).To confirm the isoform-specific promotion of PKA-C on

SRSF1-mediated tau exon 10 inclusion, we knocked down theexpression of PKA-C� or PKA-C� in SRSF1-expression cells byRNA interference and measured the splicing products of tauexon 10. Overexpression of SRSF1 promoted tau exon 10 inclu-sion. Similar to the results in Fig. 1e, down-regulation ofPKA-C� did not affect the alternative splicing of tau exon, butdown-regulation of PKA-C� increased tau exon 10 inclusion.Knockdown of the PKA-C�, but not PKA-C�, reduced the tauexon 10 inclusion by SRSF1 (supplemental Fig. 6a).To determine whether the promotion of tau exon 10 inclu-

sion by forskolin is also isoform-specific, we knocked down theexpression of PKA catalytic subunits (supplemental Fig. 1) andthen treated cells with forskolin. We found that an increase oftau exon 10 inclusion by forskolin treatment was diminished bysiPKA-C� but not by siPKA-C� (supplemental Fig. 6b). Furtherenhancement of SRSF1-promoted tau exon 10 inclusion byforskolin treatment also suppressed by siPKA-C� but notsiPKA-C� (supplemental Fig. 6b). These results further support

that PKA-C�, but not PKA-C�, promotes SRSF1 mediated tauexon 10 inclusion.Alternative splicing is regulated by splicing factors and their

phosphorylation, which results from the relative activity of thekinases and phosphatases. Cells fromdifferent sources and spe-

FIGURE 2. Activation of PKA promotes SRSF1-mediated tau exon 10 inclu-sion. HEK-293T cells were transfected with pCEP4/SRSF1-HA (a) or siRNA ofSRSF1 to knock down SRSF1 (b) for 40 h and treated with 10 �M forskolin (Fors)for 8 h. Total RNA was extracted and subjected to RT-PCR to measure tau exon10 splicing. c, SRSF1 was co-transfected with PKA-C� or C� into HEK-293FTcells for 48 h. The splicing products of tau exon 10 were determined by RT-PCR. d, two fractions of SRSF1 tagged with HA, elution 1 (E1) and E2, wereimmunopurified with anti-HA cross-linked onto protein G-agarose and sub-jected to Western blots by using anti-HA (left). Immunopurified (IP) SRSF1 inE1 and E2 was incubated with 32P-labeled RNA oligonucleotides of tau includ-ing PPE at 37 °C for 40 min. The incubation mixtures were separated by non-denaturing PAGE. The gel was dried and autoradiographed with a Phosphor-Imager. e, immunopurified SRSF1 was incubated with 32P labeled RNAoligonucleotides of tau cover PPE (tau-RNA) or deleted PPE (tau-RNA�PPE) ordeleted SC35-like element (tau-RNA�SC35-like) at 37 °C for 40 min. The incuba-tion mixture was separated by non-denaturing PAGE. The gel was dried andautoradiographed with a PhosphorImager. f, HEK-293FT cells were co-trans-fected with pCI/SI9-LI10 and pCEP4/HA or pCEP4/SRSF1-HA for 48 h, and thencells were fixed with formaldehyde, quenched with glycine, and lysed withlysis buffer. The nuclear fraction was collected, lysed, and sonicated. Theextract of the nuclear fraction was used for immunoprecipitation with anti-HA. The proteins were digested with Proteinase K in the immunoprecipitatedcomplex. The total RNA was extracted and reverse-transcribed to cDNA byusing Oligo-dT primer. Tau exon 10 of cDNA was amplified with the two setsof primers, which are described under “Experimental Procedures.” Resultsrepresent the mean � S.D.; *, p � 0.05; **, p � 0.01.



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cies and at different culture conditions have different basalactivity of splicing factors or their regulators, resulting in thedifferent splicing patterns in basal condition (Fig. 1, a, b, and c,and Fig. 2, a, b, and c) (27). Nevertheless, SRSF1 or PKA pro-motes tau exon 10 inclusion consistently.SRSF1 promotes tau exon 10 inclusion by acting on the PPE

at exon 10 (24). To confirm SRSF1 acting on PPE, we overex-pressed SRSF1-HA in the HEK-293FT cells and immunopuri-fied SRSF1with anti-HA cross-linked to proteinG-agarose.Wegot two elution fractions, E1 and E2, by elution buffer. Wefound that E1 had slower mobility than E2 on the SDS-PAGE,suggesting that phosphorylation of SRSF1 in E1was higher thanin E2 (Fig. 2d, left). Then, we incubated the purified SRSF1 with32P-labeled partial tau exon 10 RNA oligonucleotides and car-ried out EMSA.We found that SRSF1 in E1, but not in E2,madetau RNA oligonucleotides shift up (Fig. 2d, right), suggestingthat binding of SRSF1 requires certain phosphorylation.To determine whether SRSF binds onto PPE, immunopuri-

fied SRSF1 was incubated with a 32P-labeled part of tau exon 10RNA oligonucleotides (tau-RNA) containing SC35-likes andPPE elements. We found that SRSF1 slowed the mobility shiftof tau-RNA (Fig. 2e). However, deletion of PPE, but not SC35-like element, abolished the mobility shift (Fig. 2e), suggestingSRSF1 binds to the PPE of tau exon 10 mRNA.To elucidate whether PKA modulates the binding of SRSF1

to tau exon 10, we performed, using anti-HA, RNA immuno-

precipitation from pCEP4-SRSF1 and pCI/SI9-LI10 co-trans-fected HEK-293FT cells and amplified the precipitated RNAwith RT-PCR by using two sets of primers (Fig. 2f). We foundthat treatment with forskolin increased the level of taupre-mRNA co-immunoprecipitated with HA-SRSF1 by anti-HA (Fig. 2f), suggesting that PKA activation enhances the bind-ing of SRSF1 to exon 10 of tau.PKA Phosphorylates SRSF1 in Vitro and in Cultured Cells—

The biological function of SRSF1 is highly regulated by its phos-phorylation (23, 27, 34). To test whether PKA phosphorylatesSRSF1, we incubated GST-SRSF1 with PKA in vitro. Weobserved thatGST-SRSF1, but notGST,was phosphorylated byPKA in an enzyme concentration-dependent manner (Fig. 3a).To determine whether PKA phosphorylates SRSF1 in livingcells, we transfected CHO cells with pCEP4-SRSF1-HA andmetabolically labeled cells with [32P]orthophosphate. After48 h of transfection and 3 h of labeling, cells were lysed andsubjected to immunoprecipitation with anti-HA antibody.Immunoprecipitated products were separated by SDS-PAGEand visualized with phosphor-image analyzer. The resultsshowed that forskolin treatment dramatically increased phos-phorylation of SRSF1 (Fig. 3b), suggesting that PKA also phos-phorylated SRSF1 in living cells.SRSF1 has many putative phosphorylation sites. The

majority of Ser/Thr residues are located at the RS domainwith Arg-Ser repeats, which leads to difficulty in mapping

FIGURE 3. PKA phosphorylates SRSF1 in vitro and in cultured cells. a, recombinant GST-SRSF1 or GST was incubated with various concentrations of PKA inthe presence of [32P]ATP at 30 °C for 30 min, and the reaction mixture was then separated by SDS-PAGE and visualized with Coomassie blue staining (lowerpanel) or autoradiograph (upper panel). Quantitation of 32P incorporation after normalization by the protein level is shown in the graph. PSL, photostimulatedluminescence. b, CHO cells were transfected with pCEP4-SRSF1-HA for 45 h and then treated with 10 �M forskolin (Fors). At the same time, [32P]orthophosphatewas added to label the phosphoproteins. After 3 h of treatment and phospho-labeling, the cell lysates were subjected to immunoprecipitation with anti-HA.The immunoprecipitated SRSF1-HA as well as whole cell lysates was analyzed by autoradiography. c, GST fused with different deletion mutants of SRSF1 wereincubated with PKA in vitro for 30 min at 30 °C. 32P incorporation into GST-SRSF1 mutants was measured by autoradiography after the separation of thephosphorylation products by SDS-PAGE. Quantitation of the 32P incorporation after being normalized by the protein level is shown in the bar graph. Resultsrepresent the mean � S.D.; *, p � 0.05; **, p � 0.01.



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the phosphorylation sites. However, we generated severaldeletion mutants of SRSF1 fused with GST and phosphory-lated them in vitro with PKA to locate the regions of SRSF1that are phosphorylated by PKA. We observed that deletionof any domains of SRSF1, including RS (RS1�RS2) and RS2,decreased the phosphorylation by PKA but could not abolishthe phosphorylation (Fig. 3c). These results suggest that allthree regions of SRSF1, RRM, RS1, and RS2, could be phos-phorylated by PKA in vitro.PKA Interacts with SRSF1—To investigate the interaction

between PKA and various regions of SRSF1 (Fig. 4a), weperformed a GST pulldown assay and co-immunoprecipita-tion. We found that PKA-C�, but not PKA-C�, was pulleddown from rat brain extract by GST-SRSF1, but not GST(Fig. 4b), suggesting that SRSF1 interacts with PKA-C� butnot PKA-C�.To identify the regions of SRSF1 that are involved in its inter-

action with PKA-C�, we used the deletion mutants of GST-SRSF1 to pull down PKA from rat brain extract. We observedthat all SRSF1mutants pulled down PKA-C�, but not PKA-C�.Deletion of either RS domains or RRM reduced the interaction(Fig. 4b). Similar results were obtained from co-immunopre-cipitation studies in the cells treated with forskolin, but RSdomain did not co-immunoprecipitate a detectable level ofPKA-C� (Fig. 4c), suggesting that SRSF1 interacts withPKA-C� mainly through the RRM.To elucidate the interaction of SRSF1 with PKA-C� in intact

cells, we transfected pEGFP-N1-SRSF1 into HeLa cells andthen determined their subcellular localization by confocalmicroscopy.We observed that SRSF1 was localized extensivelyin the nucleus (Fig. 4d), whereas PKA-C�wasmainly located inthe cytoplasm. Forskolin treatment appeared to promote

PKA-C� translocation into the nucleus and partially co-local-ized with SRSF1 (Fig. 4d).Down-regulation of PKA Is Related to an Increase in 3R-tau

Expression inADBrain—Previously we have shown that PKA isdown-regulated in AD brain as a result of increased degrada-tion by over-activated calpain I (40). To investigate whether thedown-regulation of PKA in AD brain causes a dysregulation inthe alternative splicing of tau exon 10, wemeasured the levels of3R-tau, 4R-tau, and PKA-C� in AD brains and age- and post-mortem delay-matched normal human brains (Table 1) byWestern blot analysis. We observed that in AD brain, the totaltau level was increased by 3-fold, and 3R-tau was increasedabout 4-fold, but 4R-tau level was not significantly changed(Fig. 5, a and b), leading to an increase in the ratios of 3R-tau/total tau and 3R-tau/4R-tau (Fig. 5c). These results suggest achange in 3R-tau/4R-tau ratio and probably alternative splicingof tau exon 10 in AD brain.To study whether down-regulation of PKA-C� (Fig. 5, a and

d) correlated with the dysregulation of tau exon 10, we deter-mined levels of PKA-C� and the ratio of 3R-tau to 4R-tau inADbrain and analyzed their correlation.We found a strong inversecorrelation between the ratio of 3R-tau/4R-tau and PKA-C�levels (Fig. 5e), suggesting that the decreased PKA-C� in ADbrain might contribute to the change in the ratio of 3R-tau and4R-tau.

FIGURE 4. SRSF1 interacts with the PKA � catalytic subunit. a, variousSRSF1 deletion mutants used in this experiment are shown. b, GST-SRSF1, itsdeletion mutants, or GST coupled onto glutathione-Sepharose was incu-bated with rat brain extract, and the bound proteins were analyzed by West-ern blots developed with anti-GST, anti-PKA-C�, or anti-PKA-C�. c, SRSF1 andits deletion mutants tagged with HA were co-expressed in HEK-293FT cells for48 h. The cell extracts were immunoprecipitated with anti-HA, and the immu-noprecipitates (IP) were subjected to Western blots developed with antibod-ies indicated at the right of each blot. d, HeLa cells were transfected withGFP-SRSF1 and treated with forskolin (Fors) for 30 min followed by tripleimmunofluorescence staining. Scale bar, 10 �m.

TABLE 1Human brain tissue of AD and control (Con) cases used in this study

Case Age at death Gender PMIaBraakstageb

Tanglescoresc

Years hAD 1 89 F 3 V 14.5AD 2 80 F 2.25 VI 14.5AD 3 85 F 1.66 V 12.0AD 4 78 F 1.83 VI 15.0AD 5 95 F 3.16 VI 10.0AD 6 86 M 2.25 VI 13.5AD 7 83 F 3.00 VI 12.40AD 8 74 M 2.75 VI 14.66AD 9 79 F 1.50 VI 14.66AD 10 73 F 2.00 V 15.00AD 11 76 M 2.33 VI 15.00AD 12 72 M 2.50 VI 15.00AD 13 74 F 2.83 VI 15.00AD 14 76 M 4.00 V 15.00AD 15 78 M 1.83 VI 15.00Mean � S.D. 79.87 � 6.56 2.46 � 0.67 14.08 � 1.49Con 1 85 F 2.75 II 5.00Con 2 82 F 2.00 II 4.25Con 3 70 F 2.00 I 0.00Con 4 73 M 2.00 III 2.75Con 5 78 M 1.66 I 0.00Con 6 80 M 3.25 II 2.75Con 7 80 M 2.16 I 1.00Con 8 83 F 3.25 II 0.75Con 9 82 F 2.25 II 3.50Con 10 85 M 2.5 II 4.25Con 11 86 F 2.5 III 5.00Con 12 81 M 2.75 III 6.41Con 13 90 F 3 III 4.50Con 14 88 F 3.5 III 2.50Con 15 88 F 3 IV 4.50Mean � S.D. 82.07 � 5.48 2.57 � 0.55 3.14 � 1.98

a PMI � postmortem interval.b Neurofibrillary pathology was staged according to Braak and Braak (60).c Tangle score was a density estimate and was designated as none, sparse, moder-ate, or frequent (0, 1, 2, or 3 for statistics), as defined according to CERAD Al-zheimer disease criteria (61). Five areas (frontal, temporal, parietal, hippocam-pal, and entorhinal) were examined, and the scores were combined for amaximum of 15.



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DISCUSSION

The present study shows for the first time that the alternativesplicing of tau exon 10 is probably dysregulated in AD brain,resulting in an imbalance between the levels of 3R-tau and4R-tau. The increase in the level of 3R-tau was negatively cor-related with PKA-C� level, which is down-regulated in ADbrain due to activation of calpain I (40). Activation of PKAenhanced the binding of SRSF1 to tau pre-mRNA and pro-moted SRSF1-mediated exon 10 inclusion in cultured cells andin vivo in rat brain. PKA-C�, but not PKA-C�, interacted withand phosphorylated both the RS and RRM domains of SRSF1.Overexpression PKA-C�, but not PKA-C�, enhanced SRSF1-mediated tau exon 10 inclusion. Knockdown of PKA-C� inhib-ited SRSF1 function in promotion of tau exon 10 inclusion.Thus, down-regulation of PKA-C� in AD brain (40) might beresponsible for the increase of 3R-tau expression, resulting inan increase in 3R-tau/4R-tau ratio, which may contribute toneurofibrillary degeneration (Fig. 6).The finding of increased 3R-tau level in AD brain is inconsis-

tent with previous studies, which showed either an increase in4R-tau expression in brain regions affected by sporadic AD (15,45) or no change in tau isoforms in AD brain (13, 46). Most ofthese studies detected mRNA levels of 3R-tau and 4R-tau.However, RNA can be degraded very easily. Many studies haveshown that the postmortem interval is related to the quality ofRNA, but it is pH that affects RNA quality the most strongly. Inpostmortem tissue, the protein was found to be much morestable and its level unchanged, even when RNA was degraded(47, 48). By immunohistochemical analysis, Espinoza et al.observed that tangles appearwith both 3R-tau and 4R-tau in the

FIGURE 5. Increased ratio of 3R-tau/4R-tau in AD brain correlates with PKA-C� level. a, the levels of 3R-tau, 4R-tau, total tau, PKA-C�, and PP5 (a loadingcontrol) in the frontal cortex from six AD and six control cases were determined by Western blots developed with anti-3R-tau (RD3), anti-4R-tau (RD4), R134d,anti-PKA-C�, and anti-PP5, respectively. b, the relative levels of 3R-tau, 4R-tau, and total tau in the frontal cortex from 15 AD and 15 control cases were detectedby Western blots as described in panel a. The blots were densitometrically quantified and normalized with GAPDH. c, the relative ratios of tau proteins in thesesamples were calculated. d, the relative levels of PKA-C� in these samples were determined by Western blots developed with anti-PKA-C� and normalized withGAPDH. e, correlation of the ratio of 3R-tau/4R-tau (x axis) with PKA-C� level (y axis) is shown. The levels of PKA-C� were plotted against the ratio of 3R-tau/4R-tau in the frontal cortex from 15 AD cases. Results represent the mean � S.D.; *, p � 0.05; **, p � 0.01.

FIGURE 6. Proposed mechanism by which down-regulation of PKA con-tributes to increase in tau exon 10 exclusion and neurodegenerationvia phosphorylation of SRSF1 in AD. PKA appears to regulate tau exon10 splicing by phosphorylating SRSF1 and enhancing its binding to poly-purine enhancer cis element of exonic splicing enhancer at tau exon 10and to promote exon 10 inclusion. In AD, decreased levels of PKA due tocalpain I activation suppress its role in tau exon 10 inclusion. As a result,levels of 3R-tau increase, which disrupts the balance of the 3R-/4R-tauratio that is required for the normal function of the adult human brain andleads to aggregation of tau and the formation of neurofibrillary tangles inthe affected neurons.



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hippocampus in AD, and some advanced cases had largeamounts of thioflavin-S-positive neurofibrillary tangles onlydetected by anti-3R-tau antibody but not anti-4R-tau antibody(16). In addition, they found that the pathology appeared to bemore severe and displayedmore abundant 3R-tau-positive tan-gles in the anterior as compared with the posterior hippocam-pus from the same cases (16). These results suggest that aggre-gation and deposition of 3R-tau may be associated with moreadvanced stages. In the present study we measured the proteinlevels of 3R-tau and 4R-tau in the temporal cortices of AD andcontrol brainswith a very short postmortem interval (�3h) andfound an increase in 3R-tau level. Previously, we have demon-strated that in AD brain, overactivation of calpain I due to cal-cium dysregulation causes degradation of the regulatory sub-unit of PKA, PKA-RII (40). A decrease in PKA-RII at basalconditions provides less protection to PKA-C from degrada-tion. The C subunits of PKA are also decreased in AD brain,including C� (40), which could lead to tau exon 10 exclusion,resulting in an increase in 3R-tau expression.PKA is a Ser/Thr protein kinase and is involved in many

biological pathways. Under non-stimulated conditions, PKA ispresent as an inactive heterotetramer consisting of two C sub-units and two R subunits. There are three isoforms of C sub-units, C�, C�, and C�, and four isoforms of R subunits, RI�,RI�, RII�, and RII�. The C� isoform is ubiquitously expressed,whereas theC� isoform is expressed only in brain (43). PKA-C�is expressed only in the testis (44). Although the R� isoformsare ubiquitously expressed, the R� isoforms are predominant inthe nervous and adipose tissues. When a signal arrives at thecell surface, it activates the corresponding receptor, which inturn leads to the transient elevation of intracellular cAMP andconsequently activates PKA by dissociating the C subunits andthe R subunits. The free C subunits catalyze phosphorylation ofthe substrate proteins and then become vulnerable to degrada-tion. The present study showed differential effect of PKA-C�and PKA-C� on tau exon 10 splicing. PKA-C� promoted tauexon 10 inclusion and PKA-C� suppressed tau exon 10 inclu-sion. Further study found that PKA-C�, but not PKA-C�, inter-acted with SRSF1 and promoted SRSF1-mediated tau exon 10inclusion. The difference in sequences between PKA-C� and-C� is located in the first 50 amino acids of the N terminus,suggesting that PKA-C� via N terminus interacts with SRSF1,and PKA-C� may act on other splicing factors to inhibit tauexon 10.SRSF1harbors 2RRMmotifs and 2RSdomains and is heavily

phosphorylated in cells. There are 26 Ser, 2 Thr, and 7 Tyrputative phosphorylation sites, as predicted by using NetPhos2.0.However, themajority of Ser/Thr residues are located at theRS domain; only eight Ser/Thr residues are at the RRMdomain.Site/regional phosphorylation impacts SRSF1 function andsubcellular localization differentially (27, 34). It has beenreported that several kinases phosphorylate SRSF1 in vitro andin cultured cells. Clk and SRPK mainly phosphorylate the RSdomain and drive SRSF1 from the cytoplasm into the nucleusand from speckles into nascent transcripts, respectively (34).We recently reported that Dyrk1A phosphorylates SRSF1 atSer-227, -234, and -238 at the RS domain and leads it intospeckles (27). In the present study, by deletion mutations, we

found that PKA not only phosphorylated the RS domain butalso phosphorylated the RRM motif. The RS domain has beenshown tomediate protein-protein (49) and protein-RNA inter-actions (50), to function in nuclear import (51–53), and to playa role in the targeting of proteins such as SC35 to nuclear speck-les (54), whereas RRM determines their RNA binding specific-ity. It is known that SRSF1 binds to the PPE at exon 10 andpromotes tau exon 10 inclusion. Tau deletion mutation �K280significantly decreases the SRSF1 binding and leads to tau exon10 exclusion (24). In the present study we found that withEMSA, the hyperphosphorylated SRSF1, but not the hypophos-phorylated SRSF1, bound to oligonucleotides of tau exon 10and that with RNA immunoprecipitation assay, activation ofPKA enhanced the binding of SRSF1 binding to PPE at exon 10.These data suggest that phosphorylation of SRSF1 by PKAmight promote its binding to RNA.Although hyperphosphorylation of tau plays a fundamental

role in the development of Alzheimer-type neurofibrillarydegeneration, imbalance in the cellular levels of 3R- and 4R-tauis emerging as an important concept in this pathology. Severallines of evidence, from transgenic mouse models to humantauopathies, emphasize the importance of a critical 3R-tau/4R-tau ratio in neurons. Disturbances of the 3R-tau/4R-tau ratiomay lead to the characteristic neurofibrillary pathology. Closeto 50% of allmutations in the tau gene causing human FTDP-17affect tau exon exon 10 splicing and alter 3R/4R tau ratio (55).In addition to FTDP-17, dysregulation of exon 10 splicing mayalso contribute to other human tauopathies, such as Pick’s dis-ease (with a predominant increase in 3R-tau), progressivesupranuclear palsy (4R-tau up-regulation), corticobasal degen-eration (4R-tau up-regulation) (12), and Down syndrome (3R-tau up-regulation) (27). The neuronal tau expression levels andisoform content is highly cell- and region-specific both duringdevelopment and in the mature brain (56). 3R-tau and 4R-tauare not functionally equivalentwith respect to interactionswithmicrotubules. In vitro, 4Rhas an3-fold higher binding affinityfor microtubules than that of 3R-tau (57, 58). In addition,4R-tau is better at initiating and promotingmicrotubule assem-bly than 3R-tau (6, 59). We observed an 4-fold increase in3R-tau and an insignificant increase in 4R-tau in the AD brainwith overactivation of calpain I, resulting in an increase of3R-tau/4R-tau. Imbalanced tau apparently offers a good sub-strate for the phosphorylation and aggregation in neurons,leading to neurofibrillary degeneration.

Acknowledgments—We thank M. Marlow for editorial suggestionsand J. Murphy for secretarial assistance. The Brain Donation Pro-gram is supported in part byNIA, National Institutes of HealthGrantP30 AG19610 (to the Arizona Alzheimer’s Disease Core Center).

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Cyclic AMP-dependent Protein Kinase Regulates the Alternative Splicing of Tau Exon 10: A MECHANISM INVOLVED IN TAU PATHOLOGYOFALZHEIMER DISEASE