Ischemia induces a translocation of the splicing factor tra2-beta 1 and changes alternative splicing patterns in the brain

Ischemia Induces a T ansloca ion o he Splicing Fac o a2- ␤ 1 and Changes Al e na i e Splicing Pa e ns in he B ain Rose e Daoud, 1 Gu¨ n e Mies, 2 Aga a Smialowska, 1 Laszlo Ola´h, 1 Kons an in-Alexande Hossmann, 2 and S e an S amm 1 1 Ins i u e o Biochemis y, Uni e si y o E langen-Nu enbe g, 91054 E langen, Ge many, and 2 Max-Planck-Ins i u e o Neu ological Resea ch, 50931 Ko¨ ln, Ge many Al e na i e splice-si e selec ion is egula ed by he ela i e con- cen a ion o indi idual membe s o he se ine-a ginine amily o p o eins and he e ogeneous nuclea ibonucleop o eins. Mos o hese p o eins accumula e p edominan ly in he nucleus, and a subse o hem shu les con inuously be ween nucleus and cy osol. We demons a e ha in p ima y neu onal cul u es, a ise in in acellula calcium concen a ion induced by hapsiga - gin leads o a ansloca ion o he splicing egula o y p o ein a2- ␤ 1 and a consequen change in splice-si e selec ion. To in es iga e his phenomenon unde physiological condi ions, we used an ischemia model. Ischemia induced in he b ain causes a cy oplasmic accumula ion and hype phospho yla ion o a2- ␤ 1. In addi ion, se e al o he p o eins binding o a2- ␤ 1, such as s c associa ed in mi osis 68 and se ine/a ginine- ich p o eins, accumula e in he cy osol. Concomi an wi h his subcellula elocaliza ion, we obse ed a change in al e na i e splice-si e usage o he ICH-1 gene. The inc eased usage o i s al e na i e exons is in ag eemen wi h p e ious s udies dem- ons a ing i s ep ession by a high concen a ion o p o eins wi h se ine/a ginine- ich domains. Ou indings sugges ha a change in he calcium concen a ion associa ed wi h ischemia is pa o a signaling e en , which changes p e-mRNA splicing pa hways by causing elocaliza ion o p o eins ha egula e splice-si e selec ion. Key wo ds: al e na i e p e-mRNA p ocessing; SR p o eins; ischemia; phospho yla ion; calcium; s oke Ad ances in he human genome p ojec ha e shown ha almos all human genes con ain in ons ha a e emo ed du ing p e- mRNA p ocessing. An es ima ed 47–60% o genes con ain exons ha can be used al e na i ely (Lande e al., 2001; Mod ek e al., 2001). Al e na i e p e-mRNA p ocessing plays a key ole in gene a ing a as p o eome o 150,000–1,000,000 p o eins om he su p isingly low numbe o 30,000–40,000 genes (Lande e al., 2001; Hodges e al., 2002). Al e na i e splicing pa hways can be egula ed (e.g., du ing de elopmen ) in esponse o cellula ac i i y, in esponse o s ess, du ing p og ammed cell dea h, o as a esul o a pa hological s a e (Daoud e al., 2000; S oss e al., 2000; Akke e al., 2001; G abowski and Black, 2001; So eq and Seidman, 2001). Cu en models indica e ha a ine- uned balance o cis- elemen s and ans-ac ing ac o s is esponsible o p ope al e - na i e splice-si e selec ion (G abowski, 1998; Ellio , 2000; Smi h and Valca cel, 2000). The majo cis-elemen s comp ise 5⬘and 3⬘ splice si es and auxilia y sequence elemen s nea hem ha ac as enhance s o silence s. Those auxilia y elemen s bind o wo majo g oups o p o eins, p o eins wi h se ine-a ginine- ich domains (SR p o eins) (Fu, 1995; Manley and Tacke, 1996; G a eley, 2000) and he e ogeneous nuclea ibonucleop o eins (hnRNPs) (Weigha d e al., 1996), which can change he ecog- ni ion o splice si es because bo h SR p o eins and hnRNPs bind o componen s o he spliceosome (Tian and Mania is, 1993; He el e al., 1997; Liu e al., 1998, 2000; Chew e al., 1999). As a esul , al e na i e exons can be egula ed by modula ion o he concen a ion o SR p o eins and hnRNPs (Ca´ce es e al., 1994; Wang and Manley, 1995; Manley and Tacke, 1996) ha ha e a cha ac e is ic concen a ion in a gi en issue (Kamma e al., 1995; Hanamu a e al., 1998). S oke is a leading cause o mo bidi y and mo ali y in indus- ialized coun ies, imposing an eno mous economic bu den on he amilies o he pa ien s and he socie y o e all (Taylo e al., 1996). The igge o s oke is a ocal educ ion o blood low below he h eshold equi ed o main ain oxida i e espi a ion (Hossmann, 1994). Howe e , in he icini y o his p ima y ne- c o ic lesion, seconda y dis u bances e ol e and g adually expand and p oduce addi ional inju y, he amoun o which may ou weigh ha o he p ima y impac (Heiss e al., 1994; Gyngell e al., 1995). The easons o his delayed ischemic inju y a e only pa ly unde s ood. Gene exp ession analysis sugges s ha ⬎1000 genes a e ei he up egula ed o down egula ed by mo e han a ac o o ou , and ha many o hese may be di ec ly in ol ed in he inju y p opaga ion (T endelenbu g e al., 2000). This in eg a ed pa e n o genomic dys egula ion would also be complica ed by mis- splicing o al e na i e splicing; howe e , un il now, his ques ion has no been add essed. We demons a e ha as a eac ion o s oke, nuclea p o eins egula ing p e-mRNA splicing change hei subcellula dis ibu- ion and accumula e in he cy osol. Concomi an ly, al e na i e splice-si e selec ion o he ICH-1 gene is changed, sugges ing ha a change in al e na i e splicing pa e ns con ibu es o he ou - come o s oke. MATERIAL AND METHODS P ima y neu on cul u es. Co ex egions we e dissec ed om emb yonic day 19 a s. The issue was diges ed o 20 min wi h 500 ␮ g o papain Recei ed No . 2, 2001; e ised Ma ch 7, 2002; accep ed Ap il 19, 2002. This wo k was suppo ed by he Eu opean Union (Bio4-98-0259) and he Deu - sche Fo schungsgemeinscha (S a399/2-1 and 3/1 and SFB473/C8).We hank G ego Eichele, Anne e Ga¨ ne , and Pe e S oilo o discussions, J. Chalc o o a wo k, and Manuela Olb ich o echnical assis ance. Co espondence should be add essed o S e an S amm a he abo e add ess. E-mail: [email p o ec ed]. Copy igh © 2002 Socie y o Neu oscience 0270-6474/02/225889-11$15.00/0 The Jou nal o Neu oscience, July 15, 2002, 22(14):5889–5899 (Sigma, S . Louis, MO) in he p esence o 10 mMglucose, 1 mg/ml bo ine se um albumin, and 10 ␮ g DNase in PBS. The cells we e ca e ully dissocia ed wi h a pipe e, and he mix u e was cen i uged a 1000 ⫻g o 5 min. The cells we e esuspended in DMEM con aining 15% e al cal se um and we e pla ed on o poly-DL-o ni hine-p ecoa ed six well dishes (Nunc, Nape ille, IL). The densi y o pla ing was 1 ⫻10 6 cells/ well. App oxima ely 1 h a e pla ing, he medium was changed o a se um- ee comple e medium (S amm e al., 1993) and he cells we e cul u ed a 37°Cina10%CO 2 humidi ied a mosphe e. A e 1 week, cells we e subjec ed o ea men wi h hapsiga gin (1 ␮ M) (Sigma) o 1–24 h . Immunos aining. Fo immunohis ochemis y, C57BL/6 mice we e sub- jec ed o ansien ocal ce eb al ischemia o 1 h and he animals we e immedia ely ozen in si u in liquid ni ogen a a ious eci cula ion imes. B ains we e hen emo ed in a cold empe a u e cabine a ⫺20°C. Co onal c yos a sec ions we e cu a 20 ␮ m, placed on gela inized slides, and s o ed a ⫺20°C. Sec ions we e ixed in 4% pa a o maldehyde in PBS o 30 min and we e washed h ee imes in PBS. They we e p eincuba ed o 1 h in 3% NGS wi h 0.5% T i on X-100 in PBS a oom empe a u e and we e hen incuba ed o e nigh a 4°C wi h he a2- ␤ 1 an ise um (1:500), an i-monoclonal an ibody (mAb)104 (1:1) (Ame ican Type Cul- u e Collec ion, Manassas, VA), an i-s c associa ed in mi osis (SAM)68 (San a C uz Bio echnology, San a C uz, CA) (1:50), an i- a SAM68-like molecule-2 ( SLM-2) (1:100) (S oss e al., 2001), and an i-clea ed caspase-3 (New England Biolabs, Be e ly, MA) in PBS con aining 0.3% NGS and 0.5% T i on X-100. A e h ee washes in PBS, he sec ions we e incuba ed wi h he seconda y Cy3- luo och ome-conjuga ed goa an i- abbi o IgG mouse an ibody (Jackson ImmunoResea ch, Wes G o e, PA). Fo an i-mAb104, we used he Cy3 an i-mouse IgM an ibody a a dilu ion o 1:200 in PBS o 2 h . Nex , he sec ions we e coun e - s ained wi h 0.5 ␮ g/ml 4⬘,6-diamidino-2-phenylindole (DAPI; Sigma, Deisenho en, Ge many) in PBS o 10 min o wi h 1:200 o he nuclea Nissl coun e s ain (Neu o T ace G een Fluo escen Nissl S ain; Molec- ula P obes; Leiden, The Ne he lands), washed again h ee imes wi h PBS, and co e slipped wi h Gel-Moun (Biomeda Co po a ion, F ank- u , Ge many). Immuno luo escence images we e ob ained using con ocal lase mi- c oscopy. The gene al o e iew o one sec ion was ob ained by scanning he en i e sec ion wi h a CCD came a (Leica, Nussloch, Ge many) and a scanne in eg a ed o he mic oscope. The quan i ica ion o he a2- posi i e cells was pe o med by Neu olucida (Leica). Re e se ansc ip ion-PCR. To al RNA was ex ac ed om he s ia al egion o mice by he guanidinium hiocyana e me hod, as desc ibed p e iously (Chomczynski and Sacchi, 1987). Fo e e se ansc ip ion (RT)-PCR, cDNA was made om 1 ␮ g o o al RNA using H ⫺ -Moloney mu ine leukemia i us e e se ansc ip ase (In i ogen, San Diego, CA), 5 mM andom p ime s (P omega, Madison, WI), 0.1 mMdeoxyN- TPs, 10 U o RNasin, and 10 mMdi hio h ei ol. The eac ions we e pe o med using he ollowing p ime s: ICH e , AATTCAAGG- GACGGGTCATG; ICH o , ATGCTAACTGTCCAAGTCTA. The PCR condi ions used we e dena u a ion a 94°C o 2 min. Fo y cycles o dena u a ion (94°C o 30 sec), annealing (55°C o 30 sec), and elonga ion (72°C o 30 sec) we e hen pe o med. The inal elonga ion was pe o med a 72°C o 10 min. PCR p oduc s we e esol ed on 2% aga ose gels and we e quan i ied wi h he enhanced analysis sys em o He olab (Wiesloch, Ge many). Expe imen al g oups. Expe imen al p ocedu es we e conduc ed wi h go e nmen al app o al acco ding o he Na ional Ins i u es o Heal h guidelines o he ca e and use o labo a o y animals. Adul male C57BL/6 mice weighing 20–28 gm we e subjec ed o ansien ocal ischemia by middle ce eb al a e y (MCA) occlusion o 1 h wi hou epe usion o wi h eci cula ion o 3, 6, and 24 h (n⫽3–4 animals pe g oup). Animal su ge y. Animals we e anes he ized wi h 1% halo hane (30% O 2 , emainde N 2 O). Rec al empe a u e was main ained be ween 36.5 and 37.0°C using a eedback-con olled hea ing sys em. Du ing he expe imen s, co ical blood low was measu ed by lase Dopple low- me y (LDF) usinga1mm ibe op ic p obe (Pe imed, S ockholm, Sweden) posi ioned on he in ac skull o e he MCA e i o y o moni o LDF changes du ing ischemia and a e he onse o epe usion. Focal ce eb al ischemia was induced using an in aluminal ilamen echnique (Ha a e al., 1998). B ie ly, a midline neck incision was made and he le common and ex e nal ca o id a e ies we e isola ed and liga ed. A mic o ascula clip (FE691; Aesculap, Tu lingen, Ge many) was empo- a ily placed on he in e nal ca o id a e y. An 8-0 nylon mono ilamen (E hilon; E hicon, No de s ed , Ge many) coa ed wi h silicon esin (Xan- op en; Baye Den al, Osaka, Japan) was in oduced h ough a small incision in o he common ca o id a e y and was ad anced 9 mm dis al o he ca o id bi u ca ion o occlusion o he MCA. The size o he h ead (150–200 ␮ m) was ma ched o he body weigh o ensu e ep oducible ascula occlusion (Ha a e al., 1998). A e 60 min, epe usion was ini ia ed by wi hd awal o he h ead. Twen y minu es la e , anes hesia was discon inued and animals we e placed in o hei home cages. Ex- pe imen s we e e mina ed unde halo hane anes hesia by in si u eezing o animals. Tissue was s o ed a ⫺80°C un il addi ional p ocessing. Regional measu emen o ATP. B ains we e emo ed in a cold empe - a u e cabine (⫺20°C) and cu in o 20- ␮ m- hick c yos a sec ions. Co o- nal sec ions om he s ia al le el we e moun ed on co e slips o ATP bioluminescen imaging and on gela in-coa ed slides o immunohis o- chemis y. Fo egional ATP measu emen , co e slip-moun ed in si u ozen sec ions we e eeze-d ied and coa ed wi h a laye o ozen eac ion mix con aining he enzymes, coenzymes, and co ac o s neces- sa y o e oking ATP-speci ic bioluminescence (Kogu e and Alonso, 1978). The issue/enzyme bilaye was hawed, and ligh emission was eco ded wi h a cooled CCD came a (SensiCam) using he PC so wa e SensiCon ol (PCO CCD Imaging, Kelheim, Ge many). Wes e n blo . P o eins o immunoblo ing we e p epa ed om he s i- a al egion o con ol and ischemic hemisphe es by homogenizing 0.25 gm o issue in 1 ml o sample bu e (60 mMT is/HCl, pH 6.8, 2% SDS, 0.1 M di hio h ei ol). Boiling and cen i uga ion we e hen pe o med. P o ein (30 ␮ g) was subjec ed o SDS-PAGE (12%), as desc ibed p e iously (Laemmli, 1970), ans e ed on o ECL memb anes (Ame - sham Biosciences, A ling on Heigh s, IL), incuba ed wi h abbi a2 an ise um (Daoud e al., 1999), dilu ed 1:2000 in 1⫻NET (150 mM NaCl, 5 mM EDTA, 50 mM T is, pH 7.5, 0.05% T i on X-100, and 0.25% gela ine)/2.5⫻gela in, and de ec ed wi h an an i- abbi an ise um cou- pled o ho se adish pe oxidase (Ame sham Biosciences) (1:3000). RESULTS Blocking he sa co-endoplasma ic e iculum Ca 2ⴙ - ATPases al e s he subcellula localiza ion o he splicing egula o y p o ein a2- ␤ 1 in p ima y neu ons P e ious s udies ha e shown ha an induc ion o he mi ogen- ac i a ed p o ein kinase kinase, 38 kDa (MKK-p38) pa hway causes a cy oplasma ic accumula ion o he splicing egula o y p o eins hnRNP A1 and splicing ac o 2 (SF2)/al e na i e splic- ing ac o (ASF) ( an de Hou en an Oo d e al., 2000). We p e iously epo ed a change in splicing pa e ns a e neu onal s imula ion (Daoud e al., 1999) and wan ed o in es iga e whe he he splicing egula o y p o ein a2- ␤ 1 changes i s in a- cellula localiza ion when in acellula calcium le els a e ele- a ed. An inc ease in in acellula calcium was e oked in p ima y neu onal cul u es by blocking he sa co-endoplasma ic e iculum Ca 2⫹ -ATPases wi h hapsiga gin (T eiman e al., 1998). As shown in Figu e 1, hapsiga gin ea men causes a change in he subcellula localiza ion o endogenous a2- ␤ 1a e 1h inp i- ma y a co ical cul u es. A e 6 h , a2- ␤ 1 immuno eac i i y can no longe be de ec ed in he majo i y o nuclei (Fig. 1, 6 h ), whe eas applica ion o he sol en (DMSO) had no e ec . In hapsiga gin- ea ed cells, i could clea ly be seen ha a2- ␤ 1 immuno eac i i y was de ec able in he neu i es. Simila esul s we e ob ained when SR p o eins we e de ec ed wi h he pan- an i-SR an ibody mAb104 (da a no shown). P e ious wo k dem- ons a ed ha ac i a ion o MKK-p38 can cause a elocaliza ion o splicing ac o s. Howe e , we we e no able o de ec phospho - yla ion o MKK-p38, which would be indica i e o he ac i a ion o he MKK-p38 kinase pa hway (da a no shown). We conclude ha a change in he in acellula Ca 2⫹ concen- a ion, e oked by hapsiga gin, causes an accumula ion o a2- ␤ 1 and SR p o eins in he cy osol o p ima y neu ons. 5890 J. Neu osci., July 15, 2002, 22(14):5889–5899 Daoud e al. •Splicing and Ischemia Thapsiga gin ea men changes he al e na i e splicing pa e ns o ICH-1 in p ima y neu onal cul u es Nex , we es ed whe he he hapsiga gin-induced eloca ion o splicing ac o s changes al e na i e splicing pa e ns. Cells we e ea ed wi h hapsiga gin o 1–24 h , and he splicing pa e ns o he endogenous ICH-1 gene we e de e mined. As shown in Figu e 2, we obse ed an app oxima ely ou old inc ease in he ICH-1S o m. These da a indica e ha a change in in acellula calcium e oked by hapsiga gin can a ec p e-mRNA p ocessing pa hways. The numbe o a2- ␤ 1-posi i e nuclei dec ease in an ischemic ocus Calcium elease om in acellula s o es is he majo cause o calcium- ela ed neu onal inju y du ing ce eb al ischemia (Pas- chen e al., 1996; G ondahl e al., 1998). We he e o e asked whe he a ansloca ion o splicing ac o s is also obse ed unde such pa hological condi ions and applied an es ablished ischemia pa adigm (Ha a e al., 1996; Ha a e al., 1998). In his model, mice we e subjec ed o 60 min o su u e occlusion o he MCA and hen eci cula ion o 0, 3, 6, and 24 h . The immedia e pa hophysio- logical esponse o his ea men was a ocal deple ion o ATP ha de ines he ischemic ocus. To localize he ischemic ocus, issue sec ions we e analyzed wi h ATP-speci ic bioluminescence (Kogu e and Alonso, 1978). As expec ed, ATP was deple ed in he s ia um a he end o 1 h o MCA occlusion, bu eci cula- ion esul ed in he e u n o ATP 3 h a e ischemia. A e 24 h o eci cula ion, a ocus o seconda y ATP deple ion de eloped in Figu e 1. In acellula localiza ion o a2- ␤ 1 in p ima y neu onal cul u es a e ea men wi h hapsiga gin. P ima y co ical neu ons we e subjec ed o ea men wi h hapsiga gin. The in acellula localiza ion o a2- ␤ 1 was de e mined by immunocy ochemis y. The con ol (con) shows cells ha we e ea ed wi h DMSO only o 3 h . O he ime poin s looked simila . Le column, a2- ␤ 1 is de ec ed wi h a a2- ␤ 1 an ise um. Middle column,DAPI s aining o he same ield. Righ column, O e lay o he a2- ␤ 1 and DAPI s aining. The numbe s on he le indica e he ime o hapsiga gin ea men . Daoud e al. •Splicing and Ischemia J. Neu osci., July 15, 2002, 22(14):5889–5899 5891 he cen e o he MCA e i o y (Fig. 3A). We analyzed he exp ession o a c i ical splicing egula o y p o ein, human a2- ␤ 1 (Beil e al., 1997), a e he ischemic insul . Immunohis ochem- is y wi h an an ise um speci ic o a2- ␤ e ealed an al e ed s aining pa e n in he ischemic ocus a e 6 and 24 h o eci cula ion. In pa icula , we no iced a dec ease in he numbe o a2- ␤ 1-posi i e nuclei (Fig. 3B; see Fig. 5 o la ge magni i- ca ion). The p esence and in eg i y o he nuclei we e con i med by DAPI (Fig. 3C) and Nissl (see Fig. 5E) s aining ha also allowed us o quan i y he a2- ␤ -posi i e nuclei. We de e mined he ac ion o nuclei ha we e posi i e o a2- ␤ 1 by compa ing a2- ␤ 1 immuno eac i i y wi h he nuclea DAPI s aining, bo h in he ischemic side and in he con ala e al con ol side (Fig. 3D). As expec ed, only ⬃70% o he cells in he b ain exp essed a2- ␤ 1, which is in ag eemen wi h ou p e ious indings (Daoud e al., 1999). A e 1 h o ansien ischemia, his ac ion d opped o 50 and 40% a e 6 and 24 h o eci cula ion, espec i ely. In con as , no change in he amoun o a2- ␤ 1- posi i e nuclei was obse ed in he una ec ed con ala e al side and in co ical egions dis an o he e i o y suppo ed by he MCA. In con as , he ischemic ocus was no isible when he sec ions we e s ained o SLM-2 (S oss e al., 2001) (da a no shown) (see Fig. 6C). We conclude ha a e ansien ocal ce eb al ischemia, he numbe o a2- ␤ 1-posi i e nuclei dec eases in he cells loca ed in he ischemic ocus. Ischemia causes hype phospho yla ion o a2- ␤ 1 and a change in i s subcellula dis ibu ion Simila o o he SR p o eins, a2- ␤ 1 exis s in di e en phospho - yla ed o ms ha can be dis inguished by PAGE. We in es iga ed whe he ischemia al e s he phospho yla ion pa e n (Daoud e al., 1999). Using he ATP deple ion as a ma ke o he ischemic ocus, we isola ed issue om he ischemic ocus and he con- ala e al con ol side om adjacen sec ions and analyzed p o- ein ex ac s by Wes e n blo using an an ise um agains a2- ␤ 1. As demons a ed p e iously, he an ise um de ec s wo o ms, a slow-mig a ing hype phospho yla ed o m and a as -mig a ing hypophospho yla ed o m (Daoud e al., 1999). The ischemic insul leads o an inc ease in he hype phospho yla ed o m a e 6 and 24 h (Fig. 4). In addi ion, no change in he o al a2- ␤ 1 le el was obse ed when he a2- ␤ 1 signal was compa ed wi h ac in and his one signals (da a no shown). We hen s ained he issues wi h an an ise um agains a2- ␤ 1 and inspec ed he cells o he ischemic ocus unde highe mag- ni ica ion. Simila o he si ua ion wi h p ima y neu onal cul u e, we ound ha wi h he ischemic ocus, a2- ␤ 1 immuno eac i i y dec eased in he cell nuclei, whe eas immuno eac i i y became de ec able in he cy oplasma and he neu i es o cells (Fig. 5). These changes could be obse ed in bo h he pe iphe y and he cen e o he ocus. As expec ed, in ischemic animals wi hou eci cula ion (Fig. 5, 0 h ), a2- ␤ 1 could be de ec ed only in nuclei, whe e i was localized in a speckled pa e n. In con as , a e 6 h o eci cula ion, a2- ␤ 1 immuno eac i i y could be de ec ed in he cy oplasma su ounding he nucleus (Fig. 5, 6 h ) and he esidual nuclea s aining became mo e di use. Finally, 24 a e MCA occlusion, mos a2- ␤ 1 immuno eac i i y disap- pea ed om he nucleus and was loca ed in he su ounding cy osol (Fig. 5, 24 h ). A ha ime, a2- ␤ 1 could be de ec ed e en in neu i es eme ging om he neu ons. S aining wi h Neu o T ace Nissl con i med he in eg i y o he nuclei in cells showing a cy osolic accumula ion o a2- ␤ 1 (Fig. 5E). We conclude ha ansien ischemia induces a hype phospho- yla ion o a2- ␤ 1. This phospho yla ion occu s se e al hou s a e he ischemic insul and is accompanied by a ansloca ion o a2- ␤ 1 om he nucleus o he cy osol. A e ischemia, p o eins in e ac ing wi h a2- ␤ 1 ansloca e om he nucleus P e ious wo k shows ha p e-mRNA p ocessing occu s in a la ge mac omolecula complex in i o (Co den and Pa u ajan, 1997; McC acken e al., 1997), which has been named “ ansc ip osomal complex”o “RNA ac o y.”Se e al s udies ha e shown p e i- ously ha a2- ␤ 1 in e ac s wi h componen s o his complex, among hem he SR p o eins SRp75, SRp55, SRp40, SF2/ASF, and splicing componen 35 kDa (SC35) (Nayle e al., 1998a), he hnRNP-like p o ein sca old a achmen ac o B (Nayle e al., 1998b), he SR p o ein kinases clk1–clk4 (Nayle e al., 1997), he signal ansduc ion and ac i a ion o RNA (STAR) p o ein SLM- 2/ a2-STAR (Venables e al., 1999), and hnRNP G- ela ed p o- ein (Venables e al., 2000). Se e al SR p o eins shu le con inu- ously be ween he nucleoplasma and he cy osol, which is sugges i e o cy osolic modi ica ion o he p o eins as well as addi ional oles o hese p o eins in nuclea expo o ma u e mRNA and in ansla ion (Ca´ce es e al., 1998). In addi ion, SAM68 was shown o lea e he nucleus a e i al in ec ions (McB ide e al., 1996). We he e o e de e mined whe he a2- ␤ in e ac ing p o eins change hei subcellula localiza ion a e ischemia as well. Fi s , we used he mAb104 an ibody ha ecognizes a phos- phoepi ope p esen in all membe s o he SR p o ein amily (Neugebaue e al., 1995). As shown in Figu e 6A, mAb104 immuno eac i i y ansloca es om he nucleus o he cy osol simila ly o a2- ␤ 1. Howe e , his change in subcellula localiza- ion occu s ea lie han he one obse ed wi h a2- ␤ 1, because a signi ican numbe o cells showed mAb104 immuno eac i i y in cy osol and neu i es as ea ly as 3 h a e eoxygena ion. We subsequen ly es ed he ubiqui ously exp essed STAR p o- Figu e 2. Thapsiga gin ea men changes al e na i e splicing pa e ns. P ima y co ical neu ons we e subjec ed o ea men wi h hapsiga gin, and he splicing pa e n o he endogenous ICH-1S gene was de e mined. C, Con ol ecei ing DMSO o 3 h . D, Con ol ecei ing DMSO o 24 h . Thapsiga gin ea men imes a e indica ed a he bo om o each panel. A ep esen a i e aga ose gel o he RT-PCR p oduc s is on he le . The d awing o he igh shows schema ically he p ime localiza ion and he s uc u e o he PCR p oduc s. The s a is ical e alua ion o indepen- den expe imen s is shown he he bo om panel. E o ba s indica e he SD om a leas ou di e en expe imen s. A ows indica e he loca ion o he p ime s used o PCR. 5892 J. Neu osci., July 15, 2002, 22(14):5889–5899 Daoud e al. •Splicing and Ischemia ein amily membe SAM68 (Taylo e al., 1995; Ve ne and A z , 1997) and ound ha i also ansloca ed om he nucleus o he cy osol (Fig. 6B). Finally, we es ed he ela ed STAR p o ein amily membe SLM-2 (Di F uscio e al., 1999; S oss e al., 2001). SLM-2 egula es splice-si e selec ion by binding o pu ine- ich enhance s and was pos ula ed o be a link be ween signal- ansduc ion pa hways and p e-mRNA p ocessing (S oss e al., 2001). We ound ha his p o ein emains in he nucleus o cells in he ischemic ocus, e en 24 h a e eoxygena ion (Fig. 6C). Fu he mo e, we could nei he de ec any DNA condensa- ion in DAPI s aining, no did we obse e any abno mal Nissl s aining (Fig. 5E). Toge he , hese da a indica ed ha ischemia causes a ansloca ion o some ac o s egula ing p e-mRNA splicing bu does no a ec he nuclea in eg i y. Figu e 4. a2- ␤ 1 is hype phospho yla ed a e ischemia. Wes e n blo analysis o issue de i ed om he ischemic ocus (I) and om he una ec ed con ala e al side ha se es as a con ol (C) is shown. The eoxygena ion ime is indica ed a he op.Theopen a ow indica es he hype phospho yla ed o m, and he closed a ow poin s o he hypophos- pho yla ed o m. Molecula mass is indica ed on he le . Figu e 3. a2- ␤ 1 immuno eac i i y in an ischemic ocus. Co onal mouse b ain sec ions a e 1 h o ansien ocal ce eb al ischemia and epe usion a e shown. The columns co espond o 0, 3, 6, and 24 h a e eci cula ion. A, De e mina ion o he egional ATP con en in issue sec ions. A luci e ase assay was used. Da k a eas indica e no mal ATP le els. ATP was deple ed a e 1 h o ischemia (0 h o eci cula ion), e u ned o no mal le els a he ime o eci cula ion un il 6 h a e ischemia, and de eloped seconda y ene gy ailu e ha was clea ly isible a 24 h a e ischemia (as e isk). B, a2- ␤ 1 exp ession was de e mined in pa allel sec ions by immunohis ochemis y. Changes in he cauda e–pu amen we e al eady de ec ed a 6 h and we e clea ly e iden 24 h a e ischemia. The a ea showing elocaliza ion o a2- ␤ 1 is ma ked by a do ed line o he 24 h ime poin . Cells om his a ea a e enla ged in Figu e 5. C, DAPI s aining o a pa allel sec ion shows he in eg i y o he nuclei. D, Quan i ica ion o a2- ␤ 1-posi i e cells in he ischemic ocus (blue) and in he una ec ed co ex egions ( ed). con, A ea o con ala e al side co esponding o he ischemic ocus; IF, a ea o ischemic ocus; con, co ical egion o he con ala e al side; IS, co ical egion on he ischemic side. Daoud e al. •Splicing and Ischemia J. Neu osci., July 15, 2002, 22(14):5889–5899 5893 We hen asked whe he he changes we obse ed we e caused by cell dea h and es ed he exp ession o clea ed caspase-3, a ma ke o apop osis. As shown in Figu e 7, in he en i e ischemic ocus, only a ew cells could be de ec ed exp essing his ma ke , which is in ag eemen wi h he da a ob ained in cell cul u e. In con as , a2- ␤ 1 and SR p o ein localiza ion is s ongly a ec ed in his a ea, because app oxima ely one-hal he nuclei a e de- ple ed om a2- ␤ 1 (Fig. 3D). Fu he mo e, we did no obse e any abno mal Nissl s aining (Fig. 5E). Ou esul s indica e ha he obse ed change in subcellula localiza ion is no caused by cell Figu e 5. a2- ␤ 1 p o ein changes i s in a- cellula localiza ion a e ischemia. The isch- emic ocus a e 0, 3, 6, and 24 h o eci cu- la ion a e he occlusion, s ained wi h he a2- ␤ 1 an ise um (A–D), is shown. The cells we e aken om he pe iphe y o he isch- emic ocus. The le column shows an o e - iew o he a ec ed a ea a lowe magni ica- ion. On he igh , ep esen a i e cell nuclei a e enla ged o show he subcellula a2- ␤ 1 dis ibu ion. A he end o he 1 h ischemic in e al (0 h o eci cula ion), he speckled pa e n o a2- ␤ 1 is clea ly isible. Twen y- ou hou s a e ischemia, he p o ein is e- mo ed om he nucleus. A owheads in D indica e p o ein ha is p esen in neu i es o cells. E, Cells o he ischemic ocus a e coun- e s ained wi h Nissl s ain (cen e ) o demon- s a e he cy oplasma ic localiza ion o a2- ␤ 1 (o e lay o Nissl and a2- ␤ 1 s ain, igh ). Nissl s ain binds o RNA p esen in he nucleus. 5894 J. Neu osci., July 15, 2002, 22(14):5889–5899 Daoud e al. •Splicing and Ischemia dea h and he subsequen nec osis o he issue. Simila esul s we e seen a all o he ime poin s and wi h e minal deoxynucleo- idyl ans e ase-media ed bio inyla ed UTP nick end labeling (TUNEL) s aining (da a no shown). We conclude ha some bu no all p o eins in e ac ing wi h a2- ␤ 1 change hei subcellula localiza ion a e ansien ischemia. The al e na i e splicing pa e n o ICH-1 changes a e ischemia SR p o eins can change splice-si e selec ion in a concen a ion- dependen manne (Manley and Tacke, 1996). We in es iga ed Figu e 6 con inues. Figu e 6. In acellula localiza ion o a2- ␤ 1 in e ac ing p o eins a e ischemia. The le column shows an o e iew o he a ec ed a ea a lowe magni ica ion. The a ea analyzed is om he pe iphe y o he ischemic ocus. On he igh , ep esen a i e cell nuclei a e enla ged o show he subcellula dis ibu ion. A, De ec ion o SR p o eins wi h mAb104 in he ischemic ocus. The a owheads in A (3h) show he s aining in neu i es. B, De ec ion o SAM68 wi h an i-SAM68 an ise um in he ischemic ocus. C, De ec ion o SLM-2 in he ischemic ocus. Daoud e al. •Splicing and Ischemia J. Neu osci., July 15, 2002, 22(14):5889–5899 5895 whe he he change in nuclea SR p o ein concen a ion is con- comi an wi h a change in splice-si e selec ion. We isola ed issue om he ischemic a ea and om he con ala e al con ol side and pe o med RT-PCR. The ischemic ocus was iden i ied by he lack o ATP in adjacen sec ions (Fig. 3). We es ed he ICH-1 gene ha can gene a e wo iso o ms, ICH-1L, which p omo es apop osis, and ICH-1S, which p e en s apop osis (Wang e al., 1994), which is obse ed as a la e e ec o ischemia (Di nagl e al., 1999). Again in ag eemen wi h he si ua ion in cul u e, he ischemic episode s imula es inclusion o he al e na i e exon, which p omo es he o ma ion o he ICH-1S o m (Fig. 8). We conclude ha concomi an wi h a dec ease in nuclea concen a ion o splicing ac o s, al e na i e splice-si e selec ion is changed in an ischemic ocus. DISCUSSION P o eins egula ing p e-mRNA p ocessing change hei subcellula localiza ion a e ischemia We demons a ed ha splicing egula o y p o eins changes hei in acellula localiza ion in he b ain a e his issue has been subjec ed o ischemia. These indings a e physiologically ele an , because we elied on he analysis o endogenous p o eins in cells o in ac issue. Ou esul s a e in ag eemen wi h p e ious s udies ha used o e exp essed p o eins in ans o med cells ha we e subjec ed o osmo ic shock ( an de Hou en an Oo d e al., 2000). We obse ed elocaliza ion o a2- ␤ 1, SR p o eins, and SAM68 o he cy oplasma. Based on he mo phology, he cells whe e he ansloca ion occu ed we e mos likely neu ons. Se e al lines o e idence indica ed ha his elocaliza ion was an ac i e p ocess and no he esul o a nonspeci ic b eakdown o he nuclea en elope. Fi s , some ac o s, such as SLM-2, we e no eloca ing o he cy osol; second, ibosomal RNA complexes p esen in he nucleus did no change hei localiza ion; and hi d, no e idence o apop osis, ei he by clea ed caspase-3 o by TUNEL s aining, was obse ed. The mos likely explana ion o ou indings is ha a e ischemia, ei he he nuclea impo pa hways we e blocked o he nuclea expo was inc eased. The change in subcellula localiza ion o a2- ␤ 1 was concomi an wi h hype phospho yla ion. I emains o be es ablished whe he his change in phospho yla ion was he cause o he consequence o a change in subcellula localiza ion. Simila esul s we e ob- se ed when he in luence o cellula s ess, e oked by osmo ic shock, was s udied on hnRNP A1 ( an de Hou en an Oo d e al., 2000). Bo h a2- ␤ 1 and hnRNP A1 accumula e in he cy osol a e being hype phospho yla ed. Because bo h p o eins bind o anspo in SR and anspo in (Ka aoka e al., 1999) (ou un- published da a), a modula ion o hese sys ems by phospho yla- ion a e ischemia is an in e es ing hypo hesis ha emains o be es ed. The signal- ansduc ion pa hways leading o a change in splic- ing ac o phospho yla ion a e no clea . In con as o cellula e en s a e osmo ic shock (Ka aoka e al., 1999; an de Hou en an Oo d e al., 2000), ischemia does no ac i a e he MKK-p38 pa hway in he b ain. Blockage o he calcium eup ake in o he endoplasma ic e ic- ulum o p ima y neu onal cul u es has e ec s simila o hose o ischemia. I is well es ablished ha he cy oplasma ic calcium concen a ion inc eases a e an ischemic insul , and in he cul- u e sys em, we ound elocaliza ion o a2- ␤ 1 o he cy osol. I is he e o e likely ha se e al independen pa hways exis ha ul ima ely con e ge on he kinases ha phospho yla e hnRNP A1 and a2- ␤ 1. Splice-si e selec ion is changed a e ischemia The cu en model o al e na i e splice-si e selec ion assumes ha SR p o eins and hnRNPs o m a ne wo k ac oss he p e- mRNA ha iden i ies exons (Wu and Mania is, 1993; Manley and Tacke, 1996; He el and Mania is, 1998; S oss e al., 2000; Has - Figu e 6 con inued. 5896 J. Neu osci., July 15, 2002, 22(14):5889–5899 Daoud e al. •Splicing and Ischemia ings and K aine , 2001), which is e lec ed by hei abili y o egula e splice-si e usage in a concen a ion-dependen manne (Ca´ce es e al., 1994; Wang and Manley, 1995). The e ec o SR p o ein kinases on splice-si e selec ion has been a ibu ed o a ec ui men o SR p o eins om hei nuclea s o age si es, he speckles, and a subsequen inc ease in nuclea SR p o ein con- cen a ion (S ojdl and Bell, 1999; Mis eli, 2000). Because he eloca ion o a2- ␤ 1 and SR p o eins o he cy osol will dec ease i s concen a ion ela i e o o he ac o s egula ing splice-si e selec ion, we es ed he ICH-1 p e-mRNAs ha unde go al e na- i e splicing in he b ain. We obse ed an inc ease in inclusion o he 61 bp al e na i e exon o ICH-1. An inc ease in he SR p o ein SC35 and SF2/ASF concen a ion was shown o p omo e skipping o he 61 bp al e - na i e exon o ICH-1 (Jiang e al., 1998). The e o e, he inc ease in usage o his exon a e ischemia could be explained by he eloca ion o SR p o eins om he nucleus o he cy osol. The e ec s on RNA splicing seem o be speci ic, because he splicing pa e ns o se e al mRNAs we e no changed. Fo example, al e na i e splicing pa e ns o Bax,Bcl, and SERCA2 genes we e no a ec ed. Fu he mo e, we did no obse e an inc ease in RNA deg ada ion (da a no shown). The wo old o h ee old changes in exon usage we e compa able wi h he e ec s seen in o he sys ems ha ha e in es iga ed endogenous mRNAs (Kau e e al., 1998; Daoud e al., 1999; Xie and Black, 2001). Changes o his magni ude a e physiologically ele an ; o example, a 1.8- old inc ease in p o h ombin p e-mRNA can cause human dis- ease (Geh ing e al., 2001). In se e al sys ems s udied (e.g., d ug-induced inc ease in neu onal ac i i y and s ess e oked by o ced swimming) (Kau e e al., 1998; Daoud e al., 1999), a change in al e na i e splice-si e selec ion was ound as a molec- ula mechanism o memo ize an ex e nal s imulus. Because a2- ␤ 1 ac s on se e al p e-mRNAs con aining pu ine- ich en- hance s (P. S oilo , R. Daoud, O. Nayle , and S. S amm, unpub- lished obse a ions), i is likely ha he ansloca ion o a2- ␤ 1in cells a ec ed by ischemia will o ches a e a coo dina e change in al e na i e splice-si e selec ion o se e al genes ha will add o he long- e m e ec obse ed a e ischemia. We conclude ha cells a e able o inc ease he concen a ion o splicing egula o y p o eins by ec ui ing hem om nuclea s o - age si es and a e able o dec ease hei concen a ion in he nucleus by anspo ing hem o he cy osol. Because splice-si e selec ion is dependen on he ela i e concen a ion o splicing egula o y p o eins, p e-mRNA splicing pa e ns o some genes a e changed as a esul . Regula ion o splice-si e selec ion by subcellula localiza ion o egula o y p o eins To ou knowledge, his is he i s epo demons a ing a change in subcellula localiza ion o endogenous p o eins egula ing splice-si e selec ion in in ac issue. To de e mine possible molec- ula mechanisms, we in es iga ed whe he a change in he in a- Figu e 7. The apop o ic ma ke clea ed caspase-3 is p esen in only a ew cells in he ischemic ocus. A, DAPI s aining o a co onal sec ion, including he ischemic ocus (i.e., he le cauda e pu amen). The sec ion shown is om an animal subjec ed o 1 h o ocal ce eb al ischemia and hen 6 h o eci cula ion. B, Immunos aining o a pa allel sec ion wi h an an ise um ecognizing he clea ed caspase-3 p oduc . The box shows he a ea ha is enla ged in C.C, Enla gemen o he a ea ma ked in B. Cells exp essing clea ed caspase-3 a e indica ed wi h a owheads. Daoud e al. •Splicing and Ischemia J. Neu osci., July 15, 2002, 22(14):5889–5899 5897