Glucagon-like peptide-1 receptor is involved in learning and neuroprotection

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1 Glugon-like peptie-1 reeptor is involve in lerning n neuroprotetion Mtthew J During 1,2,Lei Co 2,Dvi S Zuzg 2,Jeremy S Frnis 1,Helen L Fitzsimons 1,2,Xingyng Jio 2, Ross J Bln 2,Mtthis Klugmnn 1,Willim A Bnks 3,Dniel J Druker 4 & Colin N Hile 2 Glugon-like peptie-1 (GLP-1) is gut peptie tht, together with its reeptor, GLP-1R, is expresse in the rin. Here we show tht intrereroventriulr (i..v.) GLP-1 n [Ser(2)]exenin(1 9) (HSEGTFTSD; homologous to onserve omin in the glugon/glp-1 fmily) enhne ssoitive n sptil lerning through GLP-1R. [Ser(2)]exenin(1 9), ut not GLP-1, is lso tive when ministere peripherlly. GLP-1R-efiient mie hve phenotype hrterize y lerning efiit tht is restore fter hippompl Glp1r gene trnsfer. In ition, rts overexpressing GLP-1R in the hippompus show improve lerning n memory. GLP-1R-efiient mie lso hve enhne seizure severity n neuronl injury fter kinte ministrtion, with n intermeite phenotype in heterozygotes n phenotypi orretion fter Glp1r gene trnsfer in hippompl somti ells. Systemi ministrtion of [Ser(2)]exenin(1 9) in wil-type nimls prevents kinte-inue poptosis of hippompl neurons. Brin GLP-1R represents promising new trget for oth ognitive-enhning n neuroprotetive gents. GLP-1 is hormone erive from tissue-speifi post-trnsltionl proessing of the proglugon gene in intestinl L ells. It shres onsierle mino i sequene homology with glugon; this sequene is onserve in multiple verterte n inverterte speies, initing n importnt role in norml physiology. Inee, GLP-1 exerts effets on gluose-epenent insulin seretion, insulin iosynthesis, gstrointestinl motility, islet -ell neogenesis, energy homeostsis n foo intke 1 4. GLP-1 n GLP-1R re lso expresse in the rin, inluing the hippompus 5 7, struture tht shows onsierle plstiity n is ruil for severl forms of lerning n memory 8. GLP-1R is ouple to multiple G-protein signl trnsution pthwys leing to tivtion of enylyl ylse, protein kinse C (PKC) n mitogentivte protein (MAP) kinse 9,1.In the rin, these pthwys re implite in plstiity n lerning n represent trgets for memory-enhning rug evelopment. We therefore hypothesize tht GLP-1R my t similrly in the rin to influene hippompl plstiity n filitte lerning. In ition, euse the hippompus is prtiulrly vulnerle to neuronl loss ssoite with epilepsy, stroke n neuroegenertive isorers, n the sme GLP-1R G-protein-ouple pthwys meite ellulr responses to poptoti stimuli, we lso explore the role of GLP-1R in neuroprotetion. RESULTS Genertion of trunte N-terminl GLP-1 nlog We investigte oth full-length GLP-1 n novel peptie, HSEGTFTSD, lso lle [Ser(2)]exenin(1 9). Bsi lol lignment serh tool nlysis revele tht these resiues re highly onserve within the GLP superfmily in oth vertertes n invertertes. These pepties inlue glugon itself n the GLP-1R gonist, exenin-4. In ontrst, N-terminlly trunte exenin(9 39) ts s GLP-1R ntgonist 11 (Fig. 1). In omprison with humn GLP-1, [Ser(2)]exenin(1 9) ws synthesize with n N-terminl steri i resiue to improve lipophiliity. A serine ws lso sustitute for glutmine in position 2 to improve peptie stility, s this resiue is ritil for ipeptiyl-peptise IV meite egrtion 12.To onfirm iologi tivity of [Ser(2)]exenin(1 9), rt insulinom ell line expressing GLP-1R (RINm5f) 13 ws inute with GLP-1 or [Ser(2)]exenin(1 9) in the presene or sene of exenin(9 39). Both GLP-1 n [Ser(2)]exenin(1 9) stimulte insulin relese, whih ws loke y exenin(9 39) (Supplementry Fig. 1 online). In fste rts, intrperitonel [Ser(2)]exenin(1 9) le to oseresponse hypoglyemi effet onsistent with gonist tivity t β-ell GLP-1R (Supplementry Fig. 2 online). Cognitive effets The effets of entrlly ministere GLP-1 n [Ser(2)]exenin(1 9) on ssoitive n sptil lerning, oth of whih re hippompl epenent, were investigte using the pssive voine 14 n Morris wter mze (MWM) prigms in rts 15. GLP-1 n [Ser(2)]exenin(1 9) ministere i..v. enhne lteny in the 1 Deprtment of Moleulr Meiine n Pthology, University of Aukln, Privte Bg 9219, Aukln 86716, New Zeln. 2 CNS Gene Therpy Center, Deprtment of Neurosurgery, Jefferson Meil College, 125 Wlnut Street, Philelphi, Pennsylvni 1917, USA. 3 GRECC, Veterns Affirs Meil Center-St. Louis n Division of Geritris, Deprtment of Internl Meiine, Sint Louis University Shool of Meiine, St. Louis, Missouri 6316, USA. 4 Deprtment of Meiine, Bnting n Best Dietes Centre, Toronto Generl Hospitl, University of Toronto, 2 Elizeth Street CCRW3-838, Toronto, Ontrio M5G 2C4, Cn. Corresponene shoul e resse to M.J.D. (m.uring@ukln..nz). Pulishe online 17 August 23; oi:1.138/nm919 NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER

2 e pssive voine tsk, similr to vsopressin (Fig. 1), peptie tht filittes lerning 16.Coinfusion of exenin(9 39) ompletely loke the memory-enhning effets of GLP-1 n [Ser(2)]exenin(1 9), ut not those of vsopressin (Fig. 1). In the MWM, rts trete i..v. with GLP-1 n [Ser(2)]exenin(1 9) trvele shorter istne to lote the pltform ompre with ontrol rts (Fig. 1). Control rts swm slightly fster thn either GLP-1- or [Ser(2)]exenin(1 9)- trete rts, ruling out extrneous motor effets (Fig. 1). The proe test showe tht oth GLP-1- n [Ser(2)]exenin(1 9)-trete rts performe etter thn vehile rts (Fig. 1e), result suggestive of enhne sptil lerning. Furthermore, enhnement of ssoitive n sptil lerning ws not ue to stress, or nxiogeni effets (Supplementry Tle 1 online) or ltere noieption (t not shown). Centrl ministrtion of rugs poses mjor prolems for trnsltion to linil pplitions. We therefore investigte the potentil of [Ser(2)]exenin(1 9) for systemi ministrtion (nsl elivery in prtiulr) 17.Intrnsl [Ser(2)]exenin(1 9), ut not GLP-1, inrese lteny in the pssive voine test to similr extent s vsopressin (Fig. 2). A srmle peptie (EDSTHFSTG), ontining the sme nine mino is s [Ser(2)]exenin(1 9) ut in rnom orer n not homologous to ny known protein, proue lteny similr to tht in vehile-trete nimls. Figure 2 Enhnement of lerning n memory y intrnsl [Ser(2)]exenin(1 9). () [Ser(2)]exenin(1 9), ut not GLP-1, enhne lteny (3 n 1 µg) in pssive voine to level omprle to tht of vsopressin (.3 µg, n = 9). +, P =.1 for [Ser(2)]exenin(1 9), 3 µg, n = 1;, P <.5 for [Ser(2)]exenin(1 9), 1 µg, n = 7 n vsopressin. Cotretment with exenin(9 39) (1 µg; n = 6) loke the effets of [Ser(2)]exenin(1 9) (1 µg) ut not of vsopressin. Vehile, n = 13; GLP-1, n = 9. () Tretments i not ffet quisition of sptil lerning. () [Ser(2)]exenin(1 9) (3 µg, n = 15) enhne retention of sptil lerning omprle to reoline (.3 mg suutneously, n = 11)., P <.1. Vehile, n = 11; GLP-1, n = 11. () The effets of [Ser(2)]exenin(1 9) (1 µg, n = 9), reoline (.3 mg, n = 5) n vsopressin (.3 µg, n = 9) on repete testing in pssive voine. [Ser(2)]exenin(1 9) enhne retention to greter egree thn i reoline or vsopressin., P <.5. Figure 1 Effets of i..v. GLP-1 n [Ser(2)]exenin(1 9) on ssoitive n sptil lerning. () Amino i sequenes of GLP superfmily. PACAP-38, pituitry enylte ylsetivting peptie; VIP, vsotive intestinl polypeptie. () Pssive voine. GLP-1 (3 ng, n =1; 1 ng, n = 6; 1 ng, n = 8;) n [Ser(2)]exenin(1 9) (3 ng, n =7; 1 ng, n =8; 1 ng, n =8) enhne lteny similr to vsopressin (3 ng, n = 4). Coinfusion of exenin(9 39) (1 ng) loke the effets of GLP-1 (n = 13) n [Ser(2)]exenin(1 9) (n = 8) ut not vsopressin (3 ng, n = 5). +, P =.1;, P <.5 ompre with vehile (n = 8) or exenin(9 39) lone (1 ng, n = 9). () Morris wter mze. GLP-1 (n = 9) n [Ser(2)]exenin(1 9) (1 ng, n =8) erese istne trvele (P <.1 ompre with vehile; n = 9) n lteny (t not shown; GLP-1, P =.1; [Ser(2)]exenin(1 9), P =.2), to fin hien pltform. Trining loks re mens of two trils per y for 5. () Both pepties erese swimming spee ompre with vehile (P <.5). (e) Proe test in the MWM., P <.5 ompre with vehile. Error rs represent s.e.m. Coministrtion of exenin(9 39) loke the ognitiveenhning effets of [Ser(2)]exenin(1 9) ut not those of vsopressin (Fig. 2). To show tht the ntgonist oul t entrlly, we riolele exenin(9 39) using 131 I, ministere it intrnslly Lteny (s) Lteny (s) Lteny (s) Vehile GLP-1 1 [Ser(2)]exenin(1 9) Areoline Exenin Exenin (9 39) (9 39) Trining trils Lteny (s) pretret Vehile Srmle peptie GLP-1 [Ser(2)]exenin(1 9) Vsopressin Exenin(9 39) Vehile GLP-1 [Ser(2)]exenin(1 9) Areoline Vehile Vsopressin Areoline [Ser(2)]exenin(1 9) 1174 VOLUME 9 NUMBER 9 SEPTEMBER 23 NATURE MEDICINE

3 to rts n mesure uptke into the olftory ul 18 n rin s well s peripherl uptke in loo n ervil lymph noes 19. Quntittion of riotivity n high-performne liqui hromtogrphy nlysis 2 showe tht intrnsl elivery of [ 131 I]exenin(9 39) resulte in effiient uptke in the olftory ul n signifint istriution in rin regions inluing the hippompus (t not shown). Clinilly pprove tretments for ognitive impirment t primrily on the holinergi system. We therefore ompre the effets of intrnsl GLP-1 n [Ser(2)]exenin(1 9) with the holinergi gonist reoline on sptil lerning in moifie version of the MWM 21. Rts were first ministere vehile, [Ser(2)]exenin(1 9), GLP-1 or reoline, n were then trine for four trils to lote sumerge pltform. They were then teste in single retention tril 48 h fter the initil trining. There were no ifferenes etween tretments s fr s quisition (Fig. 2). In ontrst, [Ser(2)]exenin(1 9) n reoline, ut not GLP-1, signifintly (P <.1) reue the ltent time for rts to lote the sumerge pltform in the retention tril (Fig. 2). Fol of Glp1r +/+ expression (.23.65) ( ) +/+ +/ / / / AAV AAV EGFP GLP-1R % freezing /+ +/ / / / / sline AAV AAV EGFP GLP-1R % ontrol lteny /+ +/ / However, intrnsl GLP-1 lowere fsting loo gluose levels wheres [Ser(2)]exenin(1 9) i not (Supplementry Tle 2 online). Disrupte gluose regultion, prtiulrly hypoglyemi, is ssoite with impire lerning 22.Moreover,GLP-1 is nxiogeni, s shown y the inrese time spent y GLP-1-trete rts in the lose rms of the elevte plus-mze (Supplementry Tle 1 online). Therefore, the nxiogeni n hypoglyemi effets of intrnsl GLP-1 my hve ompromise lerning in oth the pssive voine n MWM prigms. In light of the strong effets of [Ser(2)]exenin(1 9) on retention in the MWM, multiple tests of retention were onute using the pssive voine prigm to ompre single pretretment oses of intrnsl [Ser(2)]exenin(1 9), vsopressin n reoline. Similr lteny ws foun in ll groups t 1 n 3. At 7 fter piring, however, [Ser(2)]exenin(1 9) ws ssoite with signifintly (P <.5) greter retention thn either vsopressin or reoline (Fig. 2). Lteny (s) Sline EGFP GLP-1R Figure 3 Behviorl phenotype of wil-type Glp1r +/+, heterozygous Glp1r +/ n homozygous Glp1r / knokout mie, n resue of Glp1r / phenotype y reominnt AAV meite intrhippompl gene trnsfer of Glp1r. () Glp1r mrna expression in ge-mthe nive wil-type n knokout mie, s well s Glp1r / mie injete with AAV EGFP or AAV GLP-1R. Reltive expression level ws represente s fol of expression in Glp1r +/+ mie. Brs show verge of 4 5 mie per group, with rnge in prentheses. GLP-1R expression in oth nive Glp1r / n AAV EGFP injete mie ws elow etetion limit. () Glp1r / (n = 9) showe signifint erements in ontextul fer onitioning ompre with Glp1r +/+ mie (n = 9). Tretment with wil-type Glp1r gene resue the efiit of knokout mie. +, P <.5 for AAV GLP-1R trete mie (n = 8) ompre with sline- (n = 6) n AAV EGFP trete mie (n = 8). () [Ser(2)]exenin(1 9) enhne lteny times in Glp1r +/+ ut not Glp1r / mie in the pssive voine prigm. () AAV-meite Glp1r gene trnsfer restore the response of knokout mie to [Ser(2)]exenin(1 9) in pssive voine prigm., P <.5. e Figure 4 Overexpression of GLP-1R in rt hippompus with reominnt AAV. () Representtive rin setions showing EGFP expression in hippompus n in situ hyriiztion for Glp1r expression in nive rt n rt tht reeive AAV GLP-1R. (,) Overexpression of GLP-1R signifintly erese istne trvele to lote hien pltform in the MWM. (; P <.1) without ltering swim spee (; P >.5) () There ws no ifferene in lteny for fining visul pltform etween groups. (e) GLP-1R overexpression (n = 11) n reoline (n = 9;, P <.5) enhne freezing ehvior in ontextul fer onitioning ompre with nive (n = 9) n EGFP-trete ontrols (n = 9). Behviorl phenotype of GLP-1R-efiient mie GLP-1R-efiient mie hve mil fsting hyperglyemi n norml neuroenorine responses, ut ompletely norml feeing ehvior, fertility n generl tivity 23.We evlute ge-mthe Glp1r +/+ wil-type, Glp1r +/ heterozygous n Glp1r / knokout mie. In ition, we generte reominnt eno-ssoite virus (AAV) vetors expressing GLP-1R n enhne green fluoresent protein (EGFP), n rnomize group of the Glp1r / mie to reeive either AAV GLP-1R or AAV EGFP. We use quntittive rel-time RT-PCR to nlyze the reltive expression of Glp1r in mrna isolte from the hippompi of the five groups of mie (Glp1r +/+, Glp1r +/, nive Glp1r /,AAV EGFP trete Glp1r / n AAV GLP-1R trete Glp1r / ; Fig. 3). Compre with wil-type mie, Glp1r mrna expression in heterozygotes ws signifintly (P <.5) reue n ws unetetle in oth the nive knokout homozygotes n those tht reeive the EGFP vetor. In ontrst, the knokout homozygotes tht reeive the AAV GLP-1R vetor h hippompl Glp1r mrna levels omprle to those of wiltype mie (Fig. 3). We teste the mie in hippompl-epenent ontextul fer onitioning prigm y pling them in hmer, monitoring freezing ehvior n ministering mil shok. The next y, they were ple in the sme hmer n freezing NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER

4 Figure 5 Kini i neurotoxiity in Glp1r knokout mie n effets of intrnsl [Ser(2)]exenin(1 9) in rts. () Lteny to seizure onset ws lower in Glp1r / mie (n = 1) ompre with Glp1r +/+ mie (n = 9) n Glp1r +/ mie (n = 5). Glp1r gene trnsfer enhne resistne to kini i., P <.5;, P <.5 for AAV GLP-1R (n = 5) ompre with sline (n = 4) n AAV EGFP (n = 4). () Mximl seizure severity sores were greter in response to kini i in Glp1r / ompre with Glp1r +/+ mie (, P <.1) n Glp1r +/ mie (, P <.2). +, P <.1 for AAV GLP-1R ompre with sline n EGFP. () Representtive Fluoroje-B-stine rin setions from Glp1r +/+, Glp1r +/, Glp1r / n Glp1r / with AAV GLP-1R injetion. Less ell eth ws oserve in Glp1r +/+ ompre with Glp1r / mie (numer of Fluoroje-B-positive ells: 15.4 ± 3.8 for Glp1r +/+, 32.8 ± 3.9 for Glp1r +/ n 37.3 ± 2.5 for Glp1r / ; P <.1 for Glp1r +/ n Glp1r / ompre with Glp1r +/+ ). () TUNEL-positive ells in CA3 su-region of hippompi from rts trete with srmle peptie or [Ser(2)]exenin(1 9), or untrete nive rts. Intrnsl [Ser(2)]exenin(1 9), ut not srmle peptie, erese the numer of TUNEL-positive ells (48.43 ± 1.37 for srmle peptie, 23. ± 7.62 for [Ser(2)]exenin(1 9) n 3.5 ± 1.6 for nive; P <.5). Re rrowhes point to TUNEL-positive ells. ehvior ws mesure gin. Compre with Glp1r +/+ mie, Glp1r / mie showe mrke erese in ontextul fer onitioning (Fig. 3). The heterozygotes h n intermeite phenotype, n the AAV GLP-1R vetor, ut not the EGFP vetor, ompletely restore lerning (Fig. 3). We lso evlute the mie in the pssive voine prigm n foun tht the loss of response to [Sr(2)]exenin(1 9) in the GLP-1R-efiient mie (Fig. 3) ws fully reverse y hippompl restortion of this reeptor through AAV-meite gene trnsfer (Fig. 3). Regulte GLP-1R expression n gene trnsfer To further investigte the puttive role of GLP-1R in lerning n memory, two groups of rts were teste in the pssive voine prigm: pretretment with [Ser(2)]exenin(1 9) or pretretment with vehile. A thir group ws shm-trine (shoke only). Immeitely fter piring, the hippompus of eh rt ws proesse n reltime quntittive RT-PCR ws use to etet hnges in Glp1r mrna. Trining (vehile pretretment) inrese Glp1r mrna ompre with shm-shoke ontrols, wheres pretretment with intrnsl [Ser(2)]exenin(1 9) erese Glp1r mrna to levels foun in shm-shoke nimls n signifintly (P <.5) lowere mrna trnsript levels ompre with vehile-trete rts (Supplementry Tle 3 online). To etermine whether inresing GLP-1R levels in the hippompus woul enhne lerning in wil-type nimls, EGFP n GLP-1R AAV vetors were stereottilly injete into rts. At three weeks, roust expression ws otine with trnsgene mrna expression in the prinipl ell groups of the hippompus (Fig. 4). A seprte group of rts trete in the sme mnner ws trine twie ily for 5 in the MWM. The GLP-1R overexpressors showe mrke enhnement in mze lerning, with reutions in oth lteny (t not shown) n istne trvele to lote the hien pltform ompre with EGFP ontrols (Fig. 4). The erese in lteny ws not ue to inrese swimming spee (Fig. 4), stress effets (Supplementry Tle 1 online) or isruption in visul uity n generl loomotion n swimming ility, euse rts from oth groups similrly lote visul pltform (Fig. 4). Next we teste ssoitive lerning using ontextul fer onitioning. GLP-1R-overexpressing rts showe similr levels of freezing ompre with reolinetrete nimls n signifintly (P <.5) greter freezing ompre with EGFP n nive ontrol rts (Fig. 4e). GLP-1R n neuroprotetion Interventions tht improve synpti plstiity my e ssoite with neuroprotetion. For exmple, oth environmentl enrihment 24 n ognitive-enhning gents 25 inrese the rin s resistne to insults. GLP-1 n exenin-4 inrese neurite outgrowth in PC-12 ells 26 n show some mil protetion ginst exitotoxi neuronl mge 27 when elivere iretly into the entrl nervous system (CNS), suggesting oth neurotrophi n neuroprotetive tivity meite through rin GLP-1R. Therefore, we investigte the effets of kini i, neurotoxin whih proues exessive hippompl exittion n ell loss, prtiulrly in the CA3 suregion when ministere systemilly, in Glp1r +/+, Glp1r +/ n Glp1r / mie, s well s Glp1r / mie rnomize to reeive hippompl sline or reominnt AAV vetors expressing EGFP or GLP-1R. Signifintly (P <.5) lower seizure lteny times were oserve in Glp1r / ompre with Glp1r +/+ or heterozygote mie, n AAV-meite Glp1r gene trnsfer ompletely restore the phenotype (Fig. 5). Moreover, seizure severity ws 1176 VOLUME 9 NUMBER 9 SEPTEMBER 23 NATURE MEDICINE

5 greter in the Glp1r / mie, with n intermeite phenotype in the heterozygotes, n gene trnsfer of the reeptor lso reuing seizure severity in the knokout mie (Fig. 5). Full sttus epileptius ws oserve in one of ten Glp1r +/+ mie, ompre with six of ten Glp1r / mie n only one of five Glp1r / mie tht reeive reominnt AAV GLP-1R. Immunohistohemil omprison of the CA3 suregion of the hippompus using Fluoroje-B, fluorohrome stin speifi for egenerting neurons 28, showe signifintly (P <.1) lower ell eth in Glp1r +/+ ompre with Glp1r / mie, with reominnt AAV GLP-1R tretment proviing signifint (P <.5) neuroprotetion (Fig. 5). These results suggest tht GLP-1R my hve role in neuroprotetion. Aitionl experiments ssesse the effets of [Ser(2)]exenin(1 9) on kini i inue poptosis in the rt. Intrnsl [Ser(2)]exenin(1 9) or srmle peptie ws followe y kini i 2 min lter. Three ys fter the insult, rins were issete n TUNEL ws use to exmine DNA egrtion in the hippompus. Compre with the srmle peptie, [Ser(2)]exenin(1 9) signifintly (P <.5) ttenute kini i inue poptosis in the CA3 region of the hippompus, s mesure y the numer of TUNEL-positive ells (Fig. 5). GLP-1 signl trnsution GLP-1 reeptors re ouple to multiple G-proteins n iverse signling pthwys inluing yli enosine monophosphte, protein kinse A, phospholipse C, phosphtiylinositol-3 kinse, PKC, MAP kinses n intrellulr C 2+ (refs. 9,1,29,3). However, the ontriutions of eh of these pthwys for the mny peripherl effets of GLP-1 remin poorly hrterize, prtiulrly those of most relevne to this stuy, tht of neuroenorine ell plstiity 31,32. However, islet ell ifferentition in response to GLP-1 is loke y speifi PKC inhiitor, initing tht MAP kinse my e the likely ownstrem effetor in this moel 9.Ofinterest, reent stuies hve shown tht the ERK/MAP kinse se ppers to e onserve n ruil pthwy meiting ognition not only in severl invertertes n vertertes, ut lso in humns 33.We therefore etermine the effets of [Ser(2)]exenin(1 9) on MAP kinse in HEK 293 ells (whih o not express GLP-1R) mok-trnsfete or trnsfete with EGFP or Figure 6 Effets of [Ser(2)]exenin(1 9) on MAP kinse pthwy. () [Ser(2)]exenin(1 9) inue phosphorylte (phospho)-map kinse in Glp1r-trnsfete HEK 293 ells ut not in EGFP- or moktrnsfete ells. (,) Intrnsl [Ser(2)]exenin(1 9) enhne MAP kinse immunoretivity in the hippompus of rts., P =.5 for ytosoli frtions n P <.5 for nuler frtions (,). () Enhnement of ssoitive lerning in the pssive voine prigm y intrnsl [Ser(2)]exenin(1 9) (n = 1) ws loke y ministrtion of PD9859 (5 µg i..v.) fter trining (Post; n = 1) ut not when given efore trining (Pre; n = 9)., P <.5;, P <.1. GLP-1R. In oth mok- n EGFP-trnsfete ells, [Ser(2)]exenin(1 9) h no effet on phosphoryltion of MAP kinse, wheres there ws mrke inution of phosphoryltion in the GLP-1R-trnsfete ells fter inution with [Ser(2)]exenin(1 9) (Fig. 6). Similrly, rts were pretrete with intrnsl [Ser(2)]exenin(1 9) or vehile, n their hippompi were issete 2 min fter tretment n proe for MAP kinse tivity. Intrnsl ministrtion of [Ser(2)]exenin(1 9) signifintly (P <.5) inrese phosphorylte MAP kinse immunoretivity in ytosoli (Fig. 6,) n nuler (Fig. 6,) frtions of hippompl smples. In ition, the enhnement of ssoitive lerning y intr-nsl [Ser(2)]exenin(1 9) ws ompletely loke when PD9859, speifi MEK inhiitor tht prevents susequent ERK/MAP kinse tivtion, ws ministere to rts immeitely fter trining in the pssive voine prigm ut not when given efore trining (Fig. 6). DISCUSSION Both GLP-1 n onserve nine-mino-i N-terminl omin of the protein, [Ser(2)]exenin(1 9), enhne ssoitive n sptil lerning, n these effets were loke y GLP-1R ntgonist. In ition, n inrese in GLP-1R expression through hippompl gene trnsfer potently enhne lerning n memory. There ws lso orresponing upregultion of GLP-1R trnsripts in response to trining in n ssoitive lerning prigm. Geneti stuies, inluing phenotypi nlysis of Glp1r +/+, Glp1r +/ n Glp1r / mie n hippompl Glp1r gene trnsfer into Glp1r / mie, showe tht efiieny of this reeptor results in erements in the quisition of ssoitive ontextul lerning n tht this lerning efiit n e reverse y hippompl Glp1r somti ell gene trnsfer. In the sene of ny onfouning motor or stress effets, these results provie ruil eviene tht GLP-1R hs role in lerning n memory. In vitro stuies showe tht the effets of [Ser(2)]exenin(1 9) on insulinom ells were omprle to those of GLP-1 n were loke y the GLP-1R ntgonist exenin(9 39). Similrly, exenin(9 39) loke the enhnement of ssoitive lerning y [Ser(2)]exenin(1 9) n GLP-1. Intrnsl [Ser(2)]exenin(1 9), ministere efore trining in the pssive voine prigm, resulte in ownregultion of GLP-1R trnsripts, initing lssil gonist effet. Systemi ministrtion of [Ser(2)]exenin(1 9) lso le to oseepenent reution in fsting loo gluose, n the peptie inrese phosphorylte MAP kinse in GLP-1Rexpressing ell line ut not in the nonexpressing prent ell line. These t suggest tht [Ser(2)]exenin(1 9) exerts its effets through GLP-1R. Intrnsl [Ser(2)]exenin(1 9), ut not the srmle peptie, filitte lerning. Moreover, GLP-1 ws intive when ministere y this route. GLP-1 potently erese fsting gluose levels when elivere intrnslly. In ontrst, [Ser(2)]exenin(1 9), t oses effetive in the pssive voine tsk, h no effet on gluose when given intrnslly, n exhiite wek ut signifint hypoglyemi tivity when ministere intrperitonelly t oses tenfol greter NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER

6 (1 µg). Moreover, intrnsl GLP-1 is nxiogeni, perhps relte to moultion of loo gluose. These peripherl metoli effets of GLP-1 my hve mske ny potentil entrl effets, s hypoglyemi is ssoite with impire lerning 22 n is nxiogeni 34.However,it is lso possile tht the effiy of intrnslly ministere [Ser(2)]exenin(1 9) ompre with tht of GLP-1 my reflet ifferentil entry into the CNS. GLP-1 penetrtes the loo-rin rrier fter intrvenous ministrtion y simple iffusion 35, ut [Ser(2)]exenin(1 9), ontining just nine mino is n steri i resiue, is likely to ross the nsl epithelium n enter the rin more effiiently thn the 3-mino-i GLP-1. We were unle to lel the nonmer, ut we were suessful in leling the signifintly lrger 31-mer ntgonist, exenin(9 39), n oserve signifint rin uptke fter intrnsl ministrtion, onsistent with previous stuies on similr lrge pepties 35,36.In ition, we emonstrte the ility of intrnslly ministere ntgonist to lok the filittion of lerning fter systemi ministrtion of the nonmer peptie. It hs een suggeste tht moleules tht filitte lerning n memory my lso help protet the CNS ginst vrious insults 25. It is therefore noteworthy tht Glp1r / mie were more suseptile to kini i inue seizures n neuronl egenertion in the hippompus thn wil-type mie, with n intermeite phenotype in the heterozygotes. Similr to our t on hippomplepenent lerning, somti ell gene trnsfer of GLP-1R using reominnt AAV le to reversl of the seizure phenotype in Glp1r / mie. Furthermore, intrnsl ministrtion of [Ser(2)]exenin(1 9), ut not srmle peptie, le to lower rtes of kini i inue poptosis in hippompl neurons. Ativtion of GLP-1R filittes ellulr repir n neogenesis in the periphery, s eviene y GLP-1-inue pnreti ell ifferentition n neogenesis 31,32. Previous stuies hve shown inrese GLP-1R expression in response to penetrting rin trum 37. Moreover, GLP-1 filittes neurite outgrowth n potentites nerve growth ftor initite ellulr ifferentition in vitro 23. Our t therefore provie further eviene tht GLP-1R signling my e n importnt pthwy in neuronl plstiity n neuroprotetion. A MAP kinse inhiitor loke the memory-enhning tivity of intrnsl [Ser(2)]exenin(1 9). In ition, [Ser(2)]exenin(1 9) inrese MAP kinse tivity in the hippompus t 2 min s well s in GLP-1R-expressing ell line in vitro. These t support moel in whih tivtion of entrl GLP-1R, y either lol infusion of the full length peptie or systemi ministrtion of [Ser(2)]exenin(1 9), tivtes the ERK/MAP kinse pthwy with nuler trnslotion of p42 MAP kinse, whih is ssoite with long-term memory 38. We hve shown tht signling through GLP-1R ontriutes to lerning n memory n lso hs neuroprotetive tions. GLP-1 n [Ser(2)]exenin(1 9) t through this reeptor pthwy to proue memory-enhning effets, similr to those oserve with ognitive-enhning gents in urrent linil use. GLP-1R my therefore e promising trget for therpeuti strtegies irete towrs neuroegenertive n ognitive isorers. METHODS Animls. Mle Sprgue-Dwley rts ( 3 g), house uner ontrolle lighting n given foo liitum, were use for ll stuies. CD-1 wil-type Glp1r +/+ mie were otine from Chrles River Lortory. Glp1r / mie were proue on Chrles River Lortory CD-1 kgroun s previously esrie 23.All mie were teste t the ge of 8 weeks. All niml experiments were rrie out in ompline with the regultions of Thoms Jefferson University. Intrnsl ministrtion. Animls were nesthetize with isofluorne n ministere peptie intrnslly 2 min efore testing (in 1% β-yloextrin; 2 4 µl totl volume per nres). Intrereroventriulr ministrtion. Rts were implnte with nnul (22-guge; Plstis One) into the left ventrile (nterior-posterior (AP).8 mm, meil-lterl (ML) 1.6 mm, orsl-ventrl (DV) 3.5 mm from ur) n llowe t lest 3 4 to reover. The pepties were infuse in totl volume of 2 µl (1 µl/min), 25 min efore trining. Pssive voine. Pssive voine experiments were onute in n pprtus (MED Assoites) onsisting of one rk hmer n one light hmer tht n e ivie y guillotine oor. The trining proeure ws exeute s previously esrie 39.Rts were ministere 1.-mA shok for 3 s; mie were ministere.5-ma shok for 5 s. Retention tests were performe t 1, 3 or 7 fter piring. Mximum lteny ws 6 s for rts n 3 s for mie. Contextul fer onitioning. Fer onitioning experiments were onute in moifie pprtus (MED Assoites) house in sounttenute uile. A fn uilt into the uile lso loke ny extrneous noise. The niml ws ple in the hmer n the ourrene of freezing ehvior ws mesure every 1 s for 2 min efore shok ws ministere (1. ma, 2 s for rts;.5 ma, 5 s for mie). Freezing ehvior ws mesure gin (for 5 min) the next y. The pprtus ws lene with 1% eti i fter onitioning of eh niml. Morris wter mze. Sptil lerning ws ssesse using the MWM 15. Informtion ws quntifie y the Wter 22 omputer progrm. For the intrnsl stuy, rts were given four trining trils in single y. A retention test ws one 48 h fter trining. Lteny to fin the hien pltform in one tril ws onsiere mesure of retention of sptil lerning. For the i..v. stuy, rts were trine two trils per y for 5. The visul pltform test ws onute fter the lst trining tril on y 5. Proe tests were one 4 fter the lst trining tril. During the proe test, the pltform ws remove from the pool n the nimls were llowe to swim for 6 s. The time eh niml spent in the qurnt of the pool where the pltform h previously een lote ws mesure. Stereotti injetion. Ault mle Sprgue-Dwley rts (25 3 g) were injete with either reominnt AAV EGFP or AAV GLP-1R vetor (3 1 9 prtiles) in 2 µl volume plus 1 µl of 2% mnnitol, ilterlly into the orsl hippompus (± 3.8 mm AP, ± 1.8 mm ML, ± 3.4 mm DV from skull). Vetors were infuse t rte of 2 nl/min using miroproessorontrolle mini-pump. Reominnt AAV vetors (5 1 8 prtiles) in 1 µl volume plus.5 µl of 2% mnnitol were ilterlly injete into the orsl hippompus of Glp1r / mie (± 2. mm AP, ± 1.5 mm ML, ± 1.5 mm DV from regm). Kini i inue seizures. Mie were ministere kini i (2 mg/kg intrperitonelly) then ple in ler ontiner n losely monitore for 4 min. An oserver lin to the genotype sore lteny to the first loni-toni seizure n mximl seizure severity oring to Rine 4 (see Supplementry Methos online for etils). Western lotting. HEK 293 ells were either mok-trnsfete or trnsfete with GLP-1R or EGFP plsmi using Lipofetmine (Invitrogen). Forty-eight hours fter trnsfetion, ells were expose to [Ser(2)]exenin(1 9) (1 nm) for 2 min efore hrvesting. Western lotting ws first one using ntioy to phosphorylte p44/42 MAP kinse (Thr22/Tyr24; 1:1,; Cell Signling). The memrne ws strippe n susequently proe using ntioy to p44/42 MAP kinse (1:1,; Cell Signling). For in vivo ssy, rts were trete with intrnsl [Ser(2)]exenin(1 9) n their hippompi were rpily extrte 2 min lter. Protein smples (n = 6 per group) were proe using ntioy to MAP kinse (1:2; New Engln Biols). Quntittion of immunoretivity ws hieve with NIH Imge 1.61 softwre (Ntionl Institutes of Helth) VOLUME 9 NUMBER 9 SEPTEMBER 23 NATURE MEDICINE

7 Sttistil nlysis. Vlues re expresse s men ± s.e.m. For pssive voine n MWM tsks, the overll signifine ws etermine using repete mesures ANOVA, n the ifferenes etween iniviul tretment groups were etermine using the Fisher post-ho tests (Sttview). One-wy ANOVA ws use to nlyze MWM proe tests, elevte plus-mze, seizure evlution, ell ounting n omprisons etween groups. Aitionl etils. See Supplementry Methos online for itionl methos inluing reominnt AAV vetor proution, quntittive RT-PCR, in situ hyriiztion, Fluoroje-B stining n TUNEL stining. Note: Supplementry informtion is ville on the Nture Meiine wesite. ACKNOWLEDGMENTS We thnk A. Brooks, L. Cunninghm, R. Horst n C. Leihtlein for sientifi n tehnil input n helpful isussions. This work ws supporte in prt y the Jefferson Fulty Fountion, Ntionl Institutes of Helth, Europen Moleulr Biology Orgniztion, New Zeln Helth Reserh Counil, New Eonomy Reserh Fun n Mrsen Fun of New Zeln. 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