Mechanism and treatment for learning and memory deficits in mouse models of Noonan syndrome

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1 Mechnism nd tretment for lerning nd memory deficits in mouse models of Noonn syndrome Yong-Seok Lee 1,2, Dn Ehninger 1,7, Miou Zhou 1, Jun-Young Oh 3, Minkyung Kng 2, Chuljung Kwk 4, Hyun-Hee Ryu 2, Deln Butz 5, Toshiyuki Arki 6, Ying Ci 1, J Blji 1,7, Yoshitke Sno 1, Christine I Nm 1, Hyong Kyu Kim 3, Bong-Kiun Kng 4, Corinn Burger 5, Benjmin G Neel 6 & Alcino J Silv 1 npg 214 Nture Americ, Inc. All rights reserved. In Noonn syndrome (NS) 3 5% of sujects show cognitive deficits of unknown etiology nd with no known tretment. Here, we report tht knock-in mice expressing either of two NS-ssocited muttions in Ptpn11, which encodes the nonreceptor protein tyrosine phosphtse Shp2, show hippocmpl-dependent impirments in sptil lerning nd deficits in hippocmpl longterm potentition (LTP). In ddition, virl overexpression of n NS-ssocited llele in dult mouse hippocmpus results in incresed seline excittory synptic function nd deficits in LTP nd sptil lerning, which cn e reversed y mitogen-ctivted protein kinse kinse (MEK) inhiitor. Furthermore, rief tretment with lovsttin reduces ctivtion of the GTPse Rs extrcellulr signl-relted kinse (Erk) pthwy in the rin nd normlizes deficits in LTP nd lerning in dult mice. Our results demonstrte tht incresed sl Erk ctivity nd corresponding seline increses in excittory synptic function re responsile for the LTP impirments nd, consequently, the lerning deficits in mouse models of NS. These dt lso suggest tht lovsttin or MEK inhiitors my e useful for treting the cognitive deficits in NS. NS, n utosoml dominnt genetic disorder with n incidence of ~1 in 2,5 live irths, is chrcterized y fcil normlities, short stture, motor dely nd crdic defects 1,2. Importntly, 3 5% of ptients with NS show cognitive deficits 3 6. Ptients with NS lso show clumsiness, motor dely, hering loss, deficits in sptil knowledge nd plnning, nd socil nd emotionl prolems 3,4,7. Ptients with NS lso show impirments in hippocmpl-dependent memory tsks 4,8,9. Germline muttions in genes involved in Rs-Erk signling, such s PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MAP2K1 (MEK1) nd CBL, hve een reported to cuse NS 1,1. Among those, muttions in PTPN11, which encodes SHP2, ccount for ~5% of NS cses 1. SHP2 is positive regultor of Rs-Erk signling 11, which hs crucil function in mny cellulr processes, including lerning nd memory 12. The PTPN11 muttions found in NS ptients re gin-of-function lleles tht upregulte this signling cscde 11, Cognitive prolems such s lerning disilities nd memory impirment re common in NS 3,5,6. However, little is known out the role of PTPN11 in synptic plsticity nd lerning nd memory in the mmmlin rin. Furthermore, there is no ville tretment for cognitive deficits ssocited with this common genetic disorder. Previous studies using NS mouse models derived y knock-in Ptpn11 muttions demonstrted tht the heterozygous knock-in mice show phenotypes similr to those found in NS ptients. These include short stture, crniofcil normlities, myeloprolifertive disese nd multiple crdic defects 14,16. Here we first tested whether NS mice hve deficits in lerning nd memory nd synptic plsticity. Then, we sked whether incresing SHP2 ctivity in dult rin ffects synptic function, LTP nd lerning nd memory. Finlly, we exmined whether it is possile to rescue the LTP nd lerning deficits of dult NS mice. RESULTS NS mice show deficits in sptil lerning nd memory To investigte the underlying mechnism of the lerning nd memory deficits ssocited with NS, we studied two lines of heterozygous knockin mice hroring gin-of-function muttions found in NS ptients 14,16 : nd Ptpn11 N38D/+. Previous studies showed tht the muttion cuses more severe deficits thn the Ptpn11 N38D/+ muttion 14,16. Becuse NS ptients show deficits in sptil function nd in memory tsks dependent on the hippocmpus 4,8,9, we tested oth Ptpn11 mutnts in the hidden-pltform version of the Morris wter mze 17. In this tsk, mice lern to use sptil cues round pool to find n escpe pltform hidden eneth the wter s surfce. After trining, memory is ssessed in proe trils wherein the pltform is removed nd mice serch the empty pool for 6 s. Ptpn11 N38D/+ mice nd wild-type () controls showed similr ltencies to find the hidden pltform during trining (Fig. 1) 1 Integrtive Center for Lerning nd Memory, Deprtments of Neuroiology, Psychitry nd Bioehviorl Sciences, Psychology nd Brin Reserch Institute, University of Cliforni Los Angeles, Los Angeles, Cliforni, USA. 2 Deprtment of Life Science, Chung-Ang University, Seoul, Kore. 3 Deprtment of Medicine nd Microiology, College of Medicine, Signling Disorder Reserch Center, Chunguk Ntionl University, Cheongju, Kore. 4 School of Biologicl Sciences, College of Nturl Sciences, Seoul Ntionl University, Seoul, Kore. 5 Deprtment of Neurology, University of Wisconsin-Mdison, Mdison, Wisconsin, USA. 6 Princess Mrgret Cncer Center, University Helth Network, Toronto, Ontrio, Cnd. 7 Present ddresses: DZNE, Germn Center for Neurodegenertive Diseses, Bonn, Germny (D.E.) nd Center for Neuroscience, Indin Institute of Science, Bnglore, Indi (J.B.). Correspondence should e ddressed to A.J.S. (silv@mednet.ucl.edu) or Y.-S.L. (yongseok@cu.c.kr). Received 27 July; ccepted 14 Octoer; pulished online 1 Novemer 214; doi:1.138/nn VOLUME 17 NUMBER 12 DECEMBER 214 nture NEUROSCIENCE

2 Figure 1 NS mice show sptil memory deficits. () Escpe ltencies of Ptpn11 N38D/+ (n = 9) nd littermtes (n = 11). () Time spent in the trget qudrnt in proe tril given fter 3 d of trining (Ptpn11 N38D/+, n = 9 mice, one-wy ANOVA, F 3,32 = 13.82;, n = 11 mice, one-wy ANOVA, F 3,4 = 48.48, P <.1). Two-wy ANOVA for qudrnt occupncy with genotype s etween-sujects fctor nd pool qudrnt s within-sujects fctor, genotype pool qudrnt interction: F 3,54 = 4.91, P =.19. (c) Ltency to the pltform in mutnts (n = 1) nd controls (n = 15) in the hidden-pltform version of the wter mze. P <.1, two-wy repeted mesures ANOVA. (d) Qudrnt occupncy for the proe tril conducted fter 3 d of trining (, n = 1 mice, one-wy ANOVA, F 3,36 = 2.29, P =.127;, n = 15 mice, one-wy ANOVA, F 3,56 = 23.51, P <.1). mice spent significntly less time in the trget qudrnt thn did mice (, ± 2.4%;, ± 2.9, men ± s.e.m.; P =.42; unpired two-tiled t-test). Two-wy ANOVA for qudrnt occupncy with genotype s etween-sujects fctor nd pool qudrnt s within-sujects fctor, genotype pool qudrnt interction: F 3,69 = 2.884, P =.419., not significnt (P >.5). Error rs represent men ± s.e.m. T, trget qudrnt; AR, djcent right; O, opposite; AL, djcent left. Ltency (s) c Ltency (s) Dys Dys Ptpn11 N38D/ Percentge time spent in qudrnt d Percentge time spent in qudrnt T AR O AL Ptpn11 N38D/+ T AR O AL npg 214 Nture Americ, Inc. All rights reserved. (repeted mesures nlysis of vrince (ANOVA), F 1,18 = 2.78, P =.167) nd hd comprle swimming speeds in proe trils (, ± 1.55 cm/s (men ± s.e.m.), n = 11 mice; Ptpn11 N38D/+, ±.82 cm/s, n = 9 mice; unpired two-tiled t-test, t =.554, P =.586). However, in proe trils Ptpn11 N38D/+ mutnts spent significntly less time thn did mice in the trget qudrnt (where the pltform ws locted) during trining (Fig. 1) (percentge of totl time in the pool:, ± 4.83%; Ptpn11 N38D/+, ± 4.3%; unpired two-tiled t-test, t = 2.421, P =.263). mice serched closer to the trget during the proe trils thn did mutnts (, ± 2.26 cm; Ptpn11 N38D/+, 4.18 ± 2.5 cm; unpired two-tiled t-test, t = 2.45, P =.247). In contrst, Ptpn11 N38D/+ mutnts performed normlly in the visile-pltform version of the wter mze (Supplementry Fig. 1), suggesting tht the Ptpn11 N38D/+ muttion does not impir visuomotor function or motivtion. After extended trining, the Ptpn11 N38D/+ mutnts reched level of performnce comprle to tht of mice in proe trils, demonstrting tht they cn cquire sptil informtion, leit t slower rte thn mice (Supplementry Fig. 2). In ddition, Ptpn11 N38D/+ mutnts showed deficits in contextul fer conditioning, nother hippocmpusdependent tsk (Supplementry Fig. 3). In greement with the greter severity of deficits ssocited with the D61G mutnt s compred with the N38D mutnt in oth mice nd humns 6,15,16,18, mice showed more severe ehviorl deficits thn Ptpn11 N38D/+ mice. In proe trils, mice did not serch selectively in the trget qudrnt (Fig. 1d) (F 3,36 = 2.29, P =.127 nd F 3,56 = 23.51, P <.1 for nd, respectively; one-wy ANOVA) nd spent more time serching frther from the pltform s former loction thn did littermtes (, ± 1.29 cm, n = 15 mice;, ± 2.14 cm, n = 1 mice; unpired two-tiled t-test, t = 2.634, P =.148). Even with dditionl trining, mice were unle to rech performnce levels (Supplementry Fig. 2). Furthermore, mice took longer to rech the pltform during trining for oth hidden- (Fig. 1c) (repeted mesures ANOVA, F 1,23 = 38.54, P <.1) nd fepsp slope (percentge of seline), control Ptpn11 N38D/+, control visile-pltform versions (Supplementry Fig. 1) of the Morris wter mze, nd they showed slower swimming speeds (, ± 1.27 cm/s, n = 1;, ±.46 cm/s, n = 15; unpired two-tiled t-test, t = 6.618, P <.1), which might hve contriuted to their longer ltencies to rech the pltform. Additionl ehviorl chrcteriztion in n open field test reveled tht mice were hypoctive (Supplementry Fig. 1). These dt demonstrte tht the ehviorl deficits of mice re not limited to sptil lerning nd memory normlities. Importntly, ptients with NS lso show neurologicl normlities other thn cognitive deficits, such s higher rtes of motor dely, clumsiness nd poor coordintion 2. NS mice show deficits in synptic plsticity Hippocmpl LTP in the Schffer collterl synpses of CA1 cells hs key function in sptil lerning nd memory 19. To identify the mechnism responsile for the lerning nd memory deficits cused y the Ptpn11 muttions, we exmined CA1 Schffer collterl LTP in Ptpn11 N38D/+ nd mice y performing extrcellulr field, TBS Ptpn11 N38D/+, TBS Time (min) fepsp slope (percentge of seline), control , control, TBS, TBS Time (min) Figure 2 NS mice show LTP deficits. () LTP induced y TBS (five ursts) in hippocmpl slices from Ptpn11 N38D/+ mice nd littermtes (, n = 6 slices from 6 mice; Ptpn11 N38D/+, n = 6 slices from 6 mice; repeted-mesures ANOVA: F 1,1 = 7.893, P =.185). () LTP induced y TBS (five ursts) in hippocmpl slices from nd mice (, n = 7 slices from 7 mice;, n = 7 slices from 6 mice; repeted-mesures ANOVA: F 1,12 = 5.828, P =.327). Field excittory postsynptic potentil (fepsp) slopes normlized to the verge seline response efore LTP induction (time ), plotted in 2-min locks. Smple trces show responses during seline (gry) nd the lst 1 min (lck) of the recording (verge of 1 recording trces). Verticl r,.5 mv; horizontl r, 4 ms. Control, no TBS. Error rs represent men ± s.e.m. nture NEUROSCIENCE VOLUME 17 NUMBER 12 DECEMBER

3 npg 214 Nture Americ, Inc. All rights reserved. Figure 3 MEK inhiition reverses deficits in lerning nd memory nd LTP induced y overexpression. () Immunohistochemistry for SHP2 expression in the hippocmpus of rins injected with AAV- (left) nd AAV- (right). () overexpression increses sl Erk ctivity (p-erk) nd prevents further Erk ctivtion in response to TBS. Left, representtive immunolot (out of five) showing p-erk (top) nd totl Erk (ottom) in -expressing slices nd -expressing slices (n = 5 hippocmpl slices from 5 mice per group). Slices were prepred 1 h fter TBS. (Full-length lots nd gels re presented in Supplementry Fig. 11.) Right, normlized p-erk levels (men ± s.e.m.). CTL, no TBS. P <.5. (c) reverses sptil memory deficits in - expressing mice in the Morris wter mze. -expressing mice treted with vehicle showed no preference for the trget qudrnt (Dunnett s multiple comprison fter one-wy ANOVA, P >.5, P <.5, P <.1) nd spent significntly less time in the trget qudrnt thn mice expressing treted with vehicle (unpired two-tiled t-test, t = 2.231, P =.367). Compred with vehicle, tretment significntly incresed the time spent in the trget qudrnt in -expressing mice (, ± 3.5%, n = 1, unpired two-tiled t-test, t = 2.335, P =.313). (d) overexpression in the hippocmpus impirs memory in the oject-plce recognition test. recordings in cute hippocmpl slices. Smples from Ptpn11 N38D/+ nd mice showed no sustntil differences in sl synptic trnsmission or pired-pulse fcilittion (PPF) (Supplementry Fig. 4). However, LTP induced with thet-urst stimultion (TBS; two or five thet ursts) ws significntly reduced in Ptpn11 N38D/+ mice (Fig. 2 nd Supplementry Fig. 5) (lst 1 min of recording,, ± 4.23%, n = 6 slices from 6 mice; Ptpn11 N38D/+, ± 4.81%, n = 6 slices from 6 mice; unpired two-tiled t-test, t = 2.56, P =.311). Consistent with the hypothesis tht these LTP deficits ccount for the lerning impirments in Ptpn11 mutnt mice, mice, which hve more severe lerning impirments, lso showed more severe LTP deficits thn did Ptpn11 N38D/+ mice (Fig. 2) (lst 1 min of recording,, ± 8.41%, n = 7 slices from 7 mice;, 11.8 ± 6.3%, n = 7 slices from 6 mice; unpired two-tiled t-test, t = 2.698, P =.194). As in Ptpn11 N38D/+ mice, sl synptic trnsmission nd PPF were norml in mutnts (Supplementry Fig. 4). impirs LTP nd memory in dult mice The muttions in Ptpn11 knock-in mice re present throughout development, ffect the entire ody nd could disrupt the function of rin structures other thn the hippocmpus. Similrly, NS is systemic c Percentge time spent in qudrnt f fepsp slope (percentge of seline) icle icle Control p-erk Erk T AR O AL d Explortion time (%) /vehicle / Time (min) CTL TBS D61G D61G /vehicle / New Old New Old PTPN11 developmentl disorder, nd developmentl defects re ssumed to e responsile for the cognitive deficits in these ptients 2. Virl vectors provide sptil nd temporl regultion of gene expression crucil for testing the specific role of Ptpn11 muttions in the dult mouse rin. Moreover, NS-ssocited lleles severely compromise the viility of mutnt mice 14, mking it very difficult to otin sufficient numer of mutnt mice for ll the studies we envisioned (Supplementry Tle 1). To test whether ltered Shp2 signling in the dult hippocmpus cn cuse LTP nd, consequently, lerning deficits, we overexpressed humn using recominnt deno-ssocited virus (AAV- ) in CA1, CA2 nd CA3 of the hippocmpus of dult mice. overexpression in the hippocmpus (Fig. 3 nd Supplementry Fig. 6) resulted in incresed Erk ctivtion s ssessed y immunolotting for phosphorylted Erk (p-erk), confirming tht AAV- ws functionl (Fig. 3) (n = 5 hippocmpl slices for ech group, unpired two-tiled t-test, t = 2.452, P =.398). AAV- expression impired performnce in proe trils of the wter mze (Fig. 3c). Mice expressing AAV- spent significntly less time in the trget qudrnt thn did control mice expressing AAV- (Fig. 3c) (, ± 3.38%, n = 1;, ± 2.95%, n = 13; unpired two-tiled t-test, t = 2.231, P =.367). Unlike the muttion g Potentition (%) p-erk (percentge of /CTL) e Explortion time (%) CTL TBS New Old New Old PTPN11 CTL TBS New, new loction; old, oject loction used in trining session; dshed line, no preference for ny oject. P <.5. (e) rescues memory deficits cused y overexpression s oserved in oject-plce recognition tests. P <.5. (f,g) reverses LTP deficits cused y overexpression. (f) overexpression significntly impired TBS-induced LTP, nd reversed the deficit (repeted-mesures ANOVA, F 3,72 = 14.2, P <.1). (1 µm) ws pplied for 1 h efore LTP induction nd mintined in the th throughout recording. (g) Averge percentge of field excittory postsynptic potentil (fepsp) chnges (lst 1 min of recording) in vehicle-treted AAV- ( /veh) nd AAV- (/veh) mice (AAV-, ± 4.18%, n = 7; AAV-, ± 4.38, n = 1; unpired two-tiled t-test, t = 3.625, P =.25) nd significnt reversl y tretment (146.1 ± 4.36%, n = 1; unpired two-tiled t-test, t = 2.39, P =.33). did not ffect LTP in the AAV- group (146.2 ± 4.37%, n = 7; unpired two-tiled t-test, t = 1.414, P =.183)., not significnt VOLUME 17 NUMBER 12 DECEMBER 214 nture neuroscience

4 npg 214 Nture Americ, Inc. All rights reserved. Figure 4 overexpression enhnces excittory synptic function through incresed Rs-Erk signling. () AMPA receptor medited currents were mesured t the pek of the currents (t 65 mv), nd NMDA currents were mesured 5 ms fter onset t (4 mv). The verge of 15 trces is shown. Scle rs, 1 pa nd 4 ms. () Group dt showing the incresed AMPA/NMDA current rtio in AAV- -injected mice compred with AAV- injected mice (P =.2, t = 4.754, unpired two-tiled t-test). tretment (1 µm, 1 h) significntly reversed the AMPA/NMDA current rtio in the group without ffecting -expressing mice. Two-wy ANOVA, interction etween virl tretment nd drug, F 1,31 = 1.53, P =.28. tretment hd significnt effect only on group (P =.126, t = unpired two-tiled t-test). (c) PPF rtio is unffected y. (d) overexpression increses excittory synptic function. Representtive trces of mepsc recordings from - or - expressing hippocmpus. Grphs show mepsc frequency (P =.31, F 1,3 = 1.31, two-wy ANOVA with virl tretment s etween-sujects fctor) nd mplitude (F 1, 3 =.47, P =.498, two-wy ANOVA with virl tretment s etween-sujects fctor) in AAV- nd AAV- expressing slices treted with vehicle or (1 µm). Unpired two-tiled t-test, P =.177, P =.1. Scle rs, 2 pa nd 2 ms. (e) Excittory synptic function in mice treted or untreted (vehicle) with. Representtive trces of mepsc recordings from or mice (n = 9, 9, 1 nd 9 cells for treted with vehicle, with, with vehicle nd in mice, AAV- expression did not ffect swimming speed or other performnce vriles during the cquisition phse of the wter mze (Supplementry Fig. 7), suggesting tht the cute expression of in the hippocmpus ffects only lerning nd memory, wheres deregultion of Shp2-Erk signling during development in mice might ffect other functions, including motor coordintion. Notly, overexpression of AAV PTPN11 did not ffect sl p-erk levels or sptil lerning nd memory (Supplementry Fig. 8), indicting tht the dverse impct on Erk signling nd lerning nd memory is specific to the NS-relted PTPN11 muttion. AAV- expressing mice were lso tested in oject-plce recognition, nother hippocmpus-dependent tsk; mice expressing AAV PTPN11 were used s controls (Fig. 3d). In this tsk, mice re llowed to explore two identicl ojects locted in opposite corners of the ren during trining. One of the ojects is moved to new sptil loction in the test session 24 h fter trining. Consistent with the wter mze results, expression in the CA fields of the hippocmpus cused memory deficits in this tsk: control mice spent significntly more time exploring the oject t the new loction (Fig. 3d) (n = 15 mice, ± 3.72% for new plce, one-smple pired d e f icle -vehicle - -vehicle - icle -vehicle - -vehicle - AMPA/NMDA rtio mepsc frequency (Hz) mepsc frequency (Hz) g t-test compred to 5%, t = 2.633, P =.197), ut the mice did not (Fig. 3d) (n = 15 mice, ± 4.1% for new plce, one-smple pired t-test compred to 5%, t =.636, P =.535). Importntly, AAV- expressing mice showed comprle totl explortion time to tht of control mice during trining (control, 43.7 ± 3.98 s, n = 15; AAV-, ± 4.94 s, n = 15; unpired t-test, t =.695 P =.493). Together, these dt show tht expressing in the CA fields of the hippocmpus in dult mice is sufficient to disrupt memory, nd demonstrte tht PTPN11 hs crucil role in dult rin function, in ddition to its effects on development 2. To test whether reducing Erk ctivity could reverse the memory deficits in AAV- expressing mice, we treted these mice with the MEK inhiitor or vehicle dily, 3 min efore trining. tretment (32 mg per kilogrm of ody weight, intrperitonel injection) decresed Erk ctivtion in the hippocmpus of control nd AAV- mice (Supplementry Fig. 9). This suthreshold dose of the drug does not impir sptil lerning in mice nd decreses hippocmpl Erk ctivtion in mice y only ~25% (Supplementry Fig. 9). Importntly, tretment rescued the sptil lerning deficits in mice expressing AAV- c PPF rtio mepsc mplitude (pa) mepsc mplitude (pa) Intervl (ms) with, respectively). Grphs show mepsc frequency (P =.26, two-wy ANOVA with genotype s etween-sujects fctor, F 1, 33 = 5.914) nd mplitude (two-wy ANOVA with genotype s etween-sujects fctor, F 1, 33 =.418, P =.839) in mice nd littermtes treted with (1 µm) or untreted; unpired two-tiled t-test, P <.5, P <.1. Scle rs, 2 pa nd 2 ms. (f) Representtive trces of mipsc recordings from - or -expressing hippocmpus. Scle r, 2 pa nd 1s. (g) Representtive trces of mipsc recordings from mutnt mice or littermtes. Scle rs, 2 pa nd 1 s., vehicle;, not significnt (P >.5). Error rs represent men ± s.e.m. nture NEUROSCIENCE VOLUME 17 NUMBER 12 DECEMBER

5 Figure 5 overexpression increses surfce AMPA receptor expression. (,) Representtive imges of surfce GluA1 stining in cultured neurons. lone () or nd () were co-expressed using icistronic Sindis virl vector in cultured hippocmpl neurons (DIV21)., n = 2 neurons;, n = 22 neurons. Scle r, 2 µm. (c) Representtive imges from four experiments of western lotting of totl nd iotinylted surfce proteins from cultured rt hippocmpl neurons expressing PTPN11 or. Cdherin nd R-4 were used s mrkers for surfce nd cytosol expression, respectively. (Full-length lots nd gels re presented in Supplementry Fig. 11.) (d) Surfce nd totl expression of GluA1 in neurons expressing or PTPN11. Error rs, men ± s.e.m. Two-wy ANOVA followed y Bonferroni post-test: F 1,12 = 5.74, P <.5; surfce, PTPN11 versus, P <.5. GluA1 Merge Totl Surfce c d 2 GluA1 R4 Cdherin β-actin SHP2 D61G D61G Reltive intensity of GluA1 (%) GluA1 Merge PTPN11 Totl Surfce npg 214 Nture Americ, Inc. All rights reserved. without ffecting the performnce of the AAV- group (Fig. 3c; treted with, one-wy ANOVA, F 3,36 = 1.44, P =.1; trget versus other qudrnts, Dunnett s multiple comprison test, P <.5). Consistent with the wter mze results, the sme tretment lso rescued the memory deficits in the oject-plce recognition tsk (Fig. 3e) (AAV PTPN11, n = 5; ± 2.1% for new plce, two-tiled pired t-test compred to 5%, t = 4.395, P =.117; AAV-, n = 8; 59.9 ± 3.41% for new plce, two-tiled pired t-test compred to 5%, t = 2.94, P =.229). These results demonstrte tht incresed Rs-Erk signling in CA fields of dult hippocmpus contriute to the memory deficits in mice expressing AAV-. Notly, lso reversed the memory deficits of the dult mice in the Morris wter mze, showing tht normlizing Erk ctivity in dults cn reverse the ehviorl deficits even in mutnt mice with germline muttions (Supplementry Fig. 1). Next, we sked whether AAV- expression in dults lso impirs CA1 Schffer collterl LTP. As in mutnt mice, hippocmpl slices from AAV- expressing mice showed significntly reduced LTP in response to TBS tetnus (Fig. 3f,g) (, ± 4.18%, n = 7 slices from 7 mice;, ± 4.38%, n = 1 slices from 1 mice; unpired two-tiled t-test, t = 3.625, P =.25), demonstrting tht mnipulting Shp2 signling specificlly in the CA fields of the dult hippocmpus is sufficient to impir LTP. In ddition, TBS did not further ctivte Erk in AAV- expressing hippocmpi (Fig. 3) (n = 5 hippocmpi from 5 mice for ech group, unpired two-tiled t-test, t = 1.58, P =.1527). tretment, which reversed their lerning deficits, lso normlized LTP in CA1 hippocmpl slices from the AAV- expressing mice (Fig. 3f,g) (two-wy ANOVA, F 1,3 = 6.526, P =.159; Bonferroni post-test reveled significnt effect of tretment only on the group, P <.5). It is noteworthy tht sl synptic trnsmission nd pired-pulse fcilittion were not ffected y AAV- expression or tretment (Supplementry Fig. 7). Tken together, these results indicte tht deregulted Erk ctivity cuses CA1 LTP deficits, nd these deficits re responsile for the lerning nd memory impirments in mouse models of NS. increses excittory synptic function Next, we exmined the electrophysiologicl mechnism underlying the LTP impirment in AAV- expressing mice. Increses in Rs signling fcilitte AMPA receptor trfficking to the surfce memrne 21. For exmple, expression of constitutively ctive Rs enhnces AMPA receptor medited currents in hippocmpl neurons nd impirs LTP 21. Hence, we sked whether the increses in ctivted Erk ssocited with expression enhnced AMPA currents. Whole-cell voltge clmp recordings reveled tht the AMPA/NMDA current rtio ws incresed in AAV- expressing hippocmpi (Fig. 4,) (AAV-, 1.51 ±.11, n = 1 cells from 5 mice; AAV-,.96 ±.5, n = 1 cells from 5 mice, unpired two-tiled t-test, t = 4.754, P =.2). Importntly, tretment normlized the AMPA/NMDA rtio (Fig. 4,) (AAV-, 1.7 ±.11, n = 7 cells from 6 mice; AAV- treted with vehicle versus AAV- treted with, unpired two-tiled t-test, t = 2.832, P =.126). Although the PPF rtio ws unffected y AAV- (Fig. 4c) (AAV, n = 12 cells from 5 mice; AAV-, n = 11 cells from 5 mice; repeted-mesures ANOVA, F 1,21 =.1, P =.921), miniture excittory postsynptic currents (mepsc) frequency (ut not mplitude) ws enhnced y this mnipultion (Fig. 4d) (AAV-, 1.5 ±.53 Hz, n = 9 cells from 3 mice; AAV-, 4.64 ±.94 Hz, n = 9 cells from 3 mice; unpired two-tiled t-test, t = 2.923, P =.1). The incresed excittion in -expressing mice ws reversed y tretment (Fig. 4d; vehicle, 4.64 ±.94 Hz, n = 9 cells from 3 mice;, 2.2 ±.32 Hz, n = 9 cells from 5 mice; unpired two-tiled t-test, t = 2.645, P =.177). Consistently, mepsc frequency, ut not mplitude, ws significntly higher in pyrmidl neurons of mice thn in those of mice (Fig. 4e) (, 2.68 ±.55 Hz, n = 9 cells from 5 mice;, 5.71 ±.56 Hz, n = 1 cells from 3 mice; unpired two-tiled t-test, t = 3.858, P =.13). Moreover, miniture inhiitory postsynptic current (mipsc) frequency nd mplitude were unffected in oth AAV- trnsfected mice nd mutnts (mipsc frequency: AAV-, 6.91 ±.87 Hz, n = 9 cells from 4 mice; AAV-, 6.93 ± 1.15 Hz, n = 7 cells from 4 mice, unpired two-tiled t-test, t =.22, P =.983; mipsc mplitude:, ± 1.19 pa, n = 9 cells from 4 mice; AAV-, 2.9 ± 1.62 pa, n = 7 cells from 4 mice, unpired two-tiled t-test, t =.486, P =.634; mipsc frequency:, 15.6 ± 2.8, n = 7 cells from 5 mice;, ± 3.5, n = 8 cells from 5 mice, unpired two-tiled t-test, t =.683, P =.947; mipsc mplitude:, ± 3.6 pa, n = 7 cells from 5 mice;, ± 2.32 pa, n = 8 cells from 5 mice, unpired two-tiled t-test, t =.18, P =.86) (Fig. 4f,g). Importntly, s with mice expressing AAV-, the incresed excittion in mice ws reversed y tretment (Fig. 4e) (vehicle, 5.71 ±.56 Hz, n = 1 cells from 3 mice;, 2.87 ± 1.2 Hz, n = 9 cells from 3 mice; unpired two-tiled t-test, t = 2.58, P =.226), indicting tht incresed Rs-Erk signling is responsile for the enhnced excittory synptic function ssocited with the Ptpn11 D61G muttion. To test the hypothesis tht the increse in excittion cused y the muttion is due to increses in the numer of synpses with AMPA receptors, we trnsfected cultured hippocmpl neurons (21 d in vitro (DIV)) with nd leled surfce GluA1 174 VOLUME 17 NUMBER 12 DECEMBER 214 nture neuroscience

6 npg 214 Nture Americ, Inc. All rights reserved. Figure 6 sttin tretment reverses sptil lerning nd memory nd LTP deficits in mice. () Left, representtive immunolot showing p-erk (top) nd totl Erk (ottom) levels in hippocmpus from nd mice treted with vehicle or lovsttin. Hippocmpi were dissected 6 h fter the fourth dily lovsttin injection (sucutneous injections, 1 mg per kilogrm of ody weight). (Full-length lots nd gels re presented in Supplementry Fig. 11.) Right, r grph displying normlized p-erk levels (men ± s.e.m.)., n = 8 hippocmpi,, n = 7 hippocmpi, unpired two-tiled t-test, t = 3.438, P =.44. treted with vehicle, n = 7 hippocmpi, with lovsttin, n = 8 hippocmpi, unpired two-tiled t-test, t = 2.231, P =.439. () Ltency to the hidden pltform during trining sessions for nd mice, treted with lovsttin or vehicle. Two-wy ANOVA followed y Bonferroni post-test, treted with vehicle, n = 14 mice versus with vehicle, n = 11 mice, P <.1, P <.1; with lovsttin, n = 11 mice versus vehicle treted mice, + P <.5. (c) Swimming speed in treted nd untreted nd mice. Two-wy ANOVA F 1,45 = 19.79, P <.1. (d) Qudrnt occupncy nlysis for the proe AMPA receptors (Fig. 5,). We found tht the numer of surfce GluA1 receptor clusters ws significntly higher in - expressing neurons thn in -expressing controls (Fig. 5,) (GluA1 prticle numer per 1 µm:, 8.6 ±.59, n = 2 neurons, 1,432.6 µm of dendrites;, 6.76 ±.34, n = 22 neurons, 1,759.6 µm of dendrites; unpired two-tiled t-test, t = 2.763, P =.86). This result is consistent with the increse in mepsc frequency cused y. The size of GluA1 clusters, however, ws not ffected y expression (Fig. 5,) (GluA1 prticle size (µm 2 ):,.19 ±.2, n = 2 neurons, 1,432.6 µm of dendrites;,.18 ±.2, n = 22 neurons, 1,759.6 µm of dendrites; unpired two-tiled t-test, t =.319, P =.751), result consistent with the finding of norml mepsc mplitude. To quntittively nlyze the surfce expression of GluA1, cultured neurons trnsfected with PTPN11 or were surfce leled with iotin, nd the iotinylted surfce proteins were pulled down nd nlyzed (Fig. 5c,d). Wheres the totl expression levels of GluA1 were similr in neurons expressing PTPN11 nd, the surfce expression of GluA1 ws significntly higher in - expressing neurons thn in PTPN11-expressing neurons (Fig. 5c,d) (two-wy ANOVA followed y Bonferroni post-test: interction etween frction (totl/surfce) virus (PTPN11/ ), p-erk c Swimming speed (cm s 1 ) f Erk fepsp slope (percentge of seline) p-erk (% of /) d Percentge time spent in qudrnt Time (min) Ltency (s) Dy -vehicle -vehicle -lov -lov F 1,12 = 5.74, P =.342; totl, PTPN11 versus, P >.5; surfce, PTPN11 versus, P <.5). These dt support the results from the immunocytochemistry experiments showing tht expression fcilittes the surfce expression of GluA1. These results indicte tht postsynptic chnges in AMPA receptor trfficking contriute to the increse in excittory synptic function cused y the muttion. sttin rescues deficits in mice rescued the sptil lerning deficits in dult mice (Supplementry Fig. 1), suggesting tht decresing sl Erk ctivtion cn e therpeutic strtegy for lerning deficits in NS. sttin, memer of the sttin clss of drugs, which permete the lood-rin rrier nd re pproved y the US Food nd Drug Administrtion (FDA), inhiits Rs isoprenyltion required for the memrne locliztion nd iologicl ctivity of Rs 22,23. As in AAV- trnsfected mice, p-erk levels were incresed in hippocmpi (Fig. 6) (, 1. ± 7.23%, n = 8;, ± 2.87%, n = 7; unpired two-tiled t-test, t = 3.438, P =.44). sttin tretment normlized p-erk levels in hippocmpus from mice t concentrtions tht did not ffect Erk ctivtion in controls (Fig. 6) (vehicle, ± 2.87%, T AR O AL e Proximity to trget pltform (cm) g Potentition (%) vehicle -vehicle -lov -lov tril with mice treted with vehicle (n = 14 mice, Dunnett s multiple comprison test fter one-wy ANOVA) or lovsttin (n = 13 mice, Dunnett s multiple comprison test fter one-wy ANOVA) nd mice treted with vehicle (n = 11 mice, Dunnett s multiple comprison test fter one-wy ANOVA, P >.5) or lovsttin (n = 11 mice, Dunnett s multiple comprison test fter one-wy ANOVA, P <.1). Comprisons of percentge time in trget qudrnts, unpired two-tiled t-test, P <.5. T, trget; AR, djcent right; O, opposite; AL, djcent left. (e) Proximity to trget pltform in nd mice treted with lovsttin or vehicle. Unpired two-tiled t-test, vehicle treted versus vehicle treted mice, t = 2.813, P =.99; vehicle treted versus lovsttin treted mice t = 2.284, P =.335. (f) TBS-induced LTP deficits in nd mice treted with vehicle or systemic dministrtion of lovsttin (repeted-mesures ANOVA, F 3,96 = 14.38, P <.1). (g) Averge percentge fepsp chnges (lst 1 min of recordings) in nd mice treted with lovsttin or vehicle (-vehicle, ± 5.33%, n = 7 slices from 5 mice; -lovsttin, 15.7 ± 5.49%, n = 6 slices from 4 mice; -vehicle, ± 2.31%, n = 9 slices from 5 mice; -lovsttin, ± 6.88%, n = 7 slices from 6 mice; unpired two-tiled t-test, P =.31, P <.1). Error rs, men ± s.e.m.; veh, vehicle; lov, lovsttin;, not significnt. nture NEUROSCIENCE VOLUME 17 NUMBER 12 DECEMBER

7 npg 214 Nture Americ, Inc. All rights reserved. n = 7; lovsttin, ± 6.41%, n = 8; unpired two-tiled t-test, t = 2.231, P =.439). Importntly, mice treted with lovsttin performed etter (for exmple, reching the hidden pltform of the Morris mze fster) thn those treted with vehicle only (Fig. 6) (ltency to the pltform, two-wy ANOVA followed y Bonferroni post-test, treted with vehicle versus treted with vehicle, dy 3 P <.1, dy 4 7 P <.1; treted with vehicle versus treted with lovsttin, dy 5 P <.5), lthough their swimming speeds were unchnged y the tretment (Fig. 6c) ( treted with vehicle, 18.5 ±.6 cm/s, n = 14 mice; treted with vehicle, 13.6 ± 1.3 cm/s n = 11 mice; treted with lovsttin, 17.9 ±.8 cm/s, n = 13 mice; treted with lovsttin, 14.2 ± 1.1 cm/s, n = 11 mice; two-wy ANOVA with genotype s etween-sujects fctor nd drug tretment s within-sujects fctor, effect of genotype: F 1,45 = 19.79, P <.1, interction: F 1,45 =.4489, P =.56). These dt suggest tht the lerning deficits oserved in these mice re not n rtifct of their slower swimming speeds or other performnce deficits. In proe trils, lovsttin-treted mice, unlike vehicle-treted mice, showed selective serching in the trget qudrnt (Fig. 6d,e). During proe trils, lovsttin-treted mice showed lower verge proximity to the pltform site (i.e., etter performnce) thn vehicle-treted mice, indicting tht lovsttin tretment improved the performnce of mice in proe trils (Fig. 6d,e) (percentge of time spent in trget qudrnt, vehicle, ± 4.44%, n = 11 mice; lovsttin, 45.7 ± 4.43%, n = 11 mice; unpired two-tiled t-test, t = 1.947, P =.328; proximity to trget pltform, treted with vehicle, ± 2.33 cm, n = 14 mice; treted with lovsttin, ± 3.26 cm, n = 13 mice; treted with vehicle, 49.5 ± 3.15 cm, n = 11 mice; treted with lovsttin, ± 2.53 cm, n = 11 mice; unpired two-tiled t-test, treted with vehicle versus treted with vehicle, t = 2.813, P =.99; treted with vehicle versus treted with lovsttin, t = 2.284, P =.335). Importntly, the sptil lerning performnce of lovsttin-treted mice ws indistinguishle from vehicletreted mice (Fig. 6d,e). At the concentrtion used, lovsttin hd no effect on ny mesure of lerning in mice (Fig. 6d,e). Consistent with the hypothesis tht incresed Rs-Erk ctivity leds to the LTP deficits responsile for sptil lerning impirment in Ptpn11 mutnt mice, the levels of LTP induced y 5 TBS were significntly higher in mice treted with lovsttin thn in those treted with vehicle nd were indistinguishle from those in controls (Fig. 6f,g) (two-wy ANOVA with genotype s etween-sujects fctor, F 1,25 = 5.936, P =.223, Bonferroni posttest reveled significnt effect of lovsttin tretment only on group, P <.1). By contrst, lovsttin tretment hd no effect on LTP in hippocmpl slices from mice (Fig. 6f,g). Thus, lovsttin tretment cn normlize LTP deficits nd sptil lerning impirments even in dult mice. Although we cnnot exclude the possiility tht lovsttin my ffect other iologicl processes 24, our dt suggest tht lovsttin reverses sptil lerning deficits in mice y reducing Erk ctivtion nd, consequently, correcting LTP deficits. DISCUSSION Our study provides compelling evidence tht the sptil lerning nd memory deficits in mouse models of NS re cused y enhnced Rs- Erk ctivtion, which disrupts the lnce etween excittion nd inhiition nd impirs hippocmpl LTP. Furthermore, our experiments with virl vectors demonstrte tht Ptpn11 hs criticl roles not only in regulting development 2,25, ut lso in dult rin functions. Consistent with our findings, expression of the fly ortholog of SHP2 (Csw) ering gin-of-function muttions impired long-term memory in Drosophil 26. In the present study, we used two knock-in mutnt mice hroring Ptpn11 D61G or Ptpn11 N38D muttion. The D61G muttion is ssocited with oth NS nd leukemi nd shows higher enzymtic ctivity thn N38D, which is ssocited only with NS 15. Consistently, mice showed more severe deficits in LTP nd lerning thn Ptpn11 N38D/+ mice. Although sl level of p-erk ws significntly higher in the hippocmpus of mice thn in littermtes (Fig. 6), we could not detect significnt increses in sl p-erk in the hippocmpus of Ptpn11 N38D/+ mice, perhps ecuse these mice hd deficits tht were milder overll (Supplementry Fig. 9c). The ctivtion of Rs-Erk signling fcilittes AMPA receptor trfficking during LTP 21, nd norml hyperctivtion of postsynptic Erk signling impirs hippocmpl LTP nd lerning 27,28. Our findings suggest tht the muttion increses the numer of synpses with postsynptic AMPA receptors, thus occluding LTP nd therefore impiring lerning. In greement with the hypothesis tht there re more synpses with AMPA receptors in expressing neurons thn in -expressing neurons, we found tht expression increses mepsc frequency ut does not ffect the PPF rtio (Fig. 4c), form of plsticity very sensitive to chnges in presynptic function. Additionlly, expression incresed the evoked AMPA/NMDA rtios (Fig. 4,), nother oservtion consistent with the hypothesis tht the expression results in more synpses with AMPA receptors. Importntly, these oservtions were reproduced in oth AAV- trnsfected mice nd in the germline mutnts. Importntly, expression enhnced the surfce expression of GluA1 nd incresed the numer of surfce GluA1 clusters in cultured hippocmpl neurons, finding consistent with the hypothesis tht the enhncement in excittory synptic function driven y expression is cused y postsynptic mechnisms. Interestingly, deletion of Rs-Erk regultor (SynGAP) hs een reported to increse Erk signling, increse mepsc frequency nd impir LTP 27. Deregultion of Rs-Erk signling hs een ssocited with other genetic disorders, including neurofiromtosis type I (NF1), Costello syndrome, LEOPARD syndrome, crdiofciocutneous syndrome nd Legius syndrome 1,29. Among these, studies with Nf1 +/ mice, model of NF1, demonstrted tht incresed Rs signling results in incresed GABA relese (excittion is norml in these mice) tht leds to deficits in LTP nd, consequently, lerning nd memory impirments Together, these findings demonstrte tht similr ehviorl phenotypes (for exmple, sptil lerning deficits) nd even electrophysiologicl phenotypes (i.e., LTP deficits) cn e cused y different cellulr mechnisms: increses in AMPA receptors in NS mice nd increses in GABA relese in NF1 mice. Homozygous deletion of Nf1 in mouse postntl excittory neurons does not ffect synptic trnsmission or lerning 32, wheres expression of in postntl excittory neurons disrupts oth synptic trnsmission nd lerning, direct demonstrtion of the distinct roles of these two Rs signling modultors. Here we show tht postntl tretment with lovsttin, n FDApproved drug with strong sfety profile, cn reverse deficits in lerning nd memory s well s in LTP in n dult NS mouse model. A previous study showed tht lovsttin tretment cn rescue sptil lerning prolems, ttention deficits nd pre-pulse inhiition deficits in Nf1 +/ mutnt mice 22. Thus, our studies suggest tht lovsttin my lso e useful for tretment of cognitive deficits ssocited with NS VOLUME 17 NUMBER 12 DECEMBER 214 nture neuroscience

8 npg 214 Nture Americ, Inc. All rights reserved. Methods Methods nd ny ssocited references re ville in the online version of the pper. Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments The uthors would like to thnk I. Mody, T. O Dell, P. Golshni nd memers of A.J.S. s l for their comments on the mnuscript nd for vlule discussions; R. Jones nd Y. Zhou for helping with electrophysiologicl nlysis; D.Y. Ci for sttisticl dvice; nd A. Amin, H. Shn nd R. Knier for technicl support. This work ws supported y MH84315 to A.J.S., NRF-213R1A1A16766 nd NRF-213R1A3A17257 to Y.-S.L, R37 CA49132 to B.G.N nd MEST to H.K.K. B.G.N. is lso Cnd Reserch Chir, Tier 1, nd work in his l is prtilly supported y the Ontrio Ministry of Helth nd Long Term Cre nd the Princess Mrgret Cncer Foundtion. AUTHOR CONTRIBUTIONS Y.-S.L., D.E. nd A.J.S. conceptulized the reserch, designed the experiments nd wrote the mnuscript; Y.-S.L., D.E., M.Z., M.K., H.-H.R., C.K. C.I.N. nd Y.C. performed ehviorl experiments; Y.-S.L performed whole-cell ptch clmp recordings; Y.-S.L., M.Z. nd Y.S. performed LTP recording nd iochemicl nlyses; J.-Y.O. nd H.K.K. performed immunocytochemistry nd iotinyltion experiments; T.A. nd B.G.N. provided nd Ptpn11 N38D/+ founders, discussed the results nd edited the mnuscript; D.B. nd C.B. pckged virl vectors; Y. -S.L., D.E., M.Z., J.B., H.K.K. nd B.-K.K. nlyzed the dt nd discussed the results. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Trtgli, M. & Gel, B.D. Noonn syndrome nd relted disorders: genetics nd pthogenesis. Annu. Rev. Genomics Hum. Genet. 6, (25). 2. Romno, A.A. et l. Noonn syndrome: clinicl fetures, dignosis, nd mngement guidelines. Peditrics 126, (21). 3. Lee, D.A., Portnoy, S., Hill, P., Gillerg, C. & Ptton, M.A. Psychologicl profile of children with Noonn syndrome. Dev. Med. Child Neurol. 47, (25). 4. vn der Burgt, I. et l. Ptterns of cognitive functioning in school-ged children with Noonn syndrome ssocited with vriility in phenotypic expression. J. Peditr. 135, (1999). 5. Cesrini, L. et l. Cognitive profile of disorders ssocited with dysregultion of the RAS/MAPK signling cscde. Am. J. Med. Genet. A. 149A, (29). 6. Pierpont, E.I. et l. Genotype differences in cognitive functioning in Noonn syndrome. Genes Brin Behv. 8, (29). 7. Verhoeven, W., Wingermuhle, E., Egger, J., Vn der Burgt, I. & Tuinier, S. Noonn syndrome: psychologicl nd psychitric spects. Am. J. Med. Genet. A. 146A, (28). 8. Alfieri, P. et l. Long term memory profile of disorders ssocited with dysregultion of the RAS-MAPK signling cscde. Behv. Genet. 41, (211). 9. Pierpont, E.I., Tworog-Due, E. & Roerts, A.E. Lerning nd memory in children with Noonn syndrome. Am. J. Med. Genet. A 161, (213). 1. Zenker, M. Clinicl mnifesttions of muttions in RAS nd relted intrcellulr signl trnsduction fctors. Curr. Opin. Peditr. 23, (211). 11. Neel, B.G., Gu, H. & Po, L. The Shp ing news: SH2 domin-contining tyrosine phosphtses in cell signling. Trends Biochem. Sci. 28, (23). 12. Swett, J.D. The neuronl MAP kinse cscde: iochemicl signl integrtion system suserving synptic plsticity nd memory. J. Neurochem. 76, 1 1 (21). 13. Frgle, A., Trtgli, M., Wu, J. & Gel, B.D. Noonn syndrome-ssocited SHP2/ PTPN11 mutnts cuse EGF-dependent prolonged GAB1 inding nd sustined ERK2/MAPK1 ctivtion. Hum. Mutt. 23, (24). 14. Arki, T. et l. Mouse model of Noonn syndrome revels cell type nd gene dosge dependent effects of Ptpn11 muttion. Nt. Med. 1, (24). 15. Keilhck, H., Dvid, F.S., McGregor, M., Cntley, L.C. & Neel, B.G. Diverse iochemicl properties of Shp2 mutnts. Implictions for disese phenotypes. J. Biol. Chem. 28, (25). 16. Arki, T. et l. Noonn syndrome crdic defects re cused y PTPN11 cting in endocrdium to enhnce endocrdil-mesenchyml trnsformtion. Proc. Ntl. Acd. Sci. USA 16, (29). 17. Morris, R.G., Grrud, P., Rwlins, J.N. & O Keefe, J. Plce nvigtion impired in rts with hippocmpl lesions. Nture 297, (1982). 18. Trtgli, M. et l. Diversity nd functionl consequences of germline nd somtic PTPN11 muttions in humn disese. Am. J. Hum. Genet. 78, (26). 19. Lee, Y.S. & Silv, A.J. The moleculr nd cellulr iology of enhnced cognition. Nt. Rev. Neurosci. 1, (29). 2. Guthier, A.S. et l. Control of CNS cell-fte decisions y SHP-2 nd its dysregultion in Noonn syndrome. Neuron 54, (27). 21. Zhu, J.J., Qin, Y., Zho, M., Vn Aelst, L. & Mlinow, R. Rs nd Rp control AMPA receptor trfficking during synptic plsticity. Cell 11, (22). 22. Li, W. et l. The HMG-CoA reductse inhiitor lovsttin reverses the lerning nd ttention deficits in mouse model of neurofiromtosis type 1. Curr. Biol. 15, (25). 23. Seti, S.M., Tklcevic, G.T. & Jni, J.P. sttin, cholesterol iosynthesis inhiitor, inhiits the growth of humn H-rs oncogene trnsformed cells in nude mice. Cncer Commun. 3, (1991). 24. Milmn, T., Hrihrn, M. & Krten, B. Inhiition of neuronl cholesterol iosynthesis with lovsttin leds to impired synptic vesicle relese even in the presence of lipoproteins or gernylgerniol. J. Neurochem. 119, (211). 25. Lee, S.H. et l. Synpses re regulted y the cytoplsmic tyrosine kinse Fer in pthwy medited y p12ctenin, Fer, SHP-2, nd β-ctenin. J. Cell Biol. 183, (28). 26. Pgni, M.R., Oishi, K., Gel, B.D. & Zhong, Y. The phosphtse SHP2 regultes the spcing effect for long-term memory induction. Cell 139, (29). 27. Rumugh, G., Adms, J.P., Kim, J.H. & Hugnir, R.L. SynGAP regultes synptic strength nd mitogen-ctivted protein kinses in cultured neurons. Proc. Ntl. Acd. Sci. USA 13, (26). 28. Stornett, R.L. & Zhu, J.J. Rs nd Rp signling in synptic plsticity nd mentl disorders. Neuroscientist 17, (211). 29. Tidymn, W.E. & Ruen, K.A. The RASopthies: developmentl syndromes of Rs/ MAPK pthwy dysregultion. Curr. Opin. Genet. Dev. 19, (29). 3. Shilynsky, C., Lee, Y.S. & Silv, A.J. Moleculr nd cellulr mechnisms of lerning disilities: focus on NF1. Annu. Rev. Neurosci. 33, (21). 31. Shilynsky, C. et l. Neurofiromin regultes corticostritl inhiitory networks during working memory performnce. Proc. Ntl. Acd. Sci. USA 17, (21). 32. Cui, Y. et l. Neurofiromin regultion of ERK signling modultes GABA relese nd lerning. Cell 135, (28). 33. Cost, R.M. et l. Mechnism for the lerning deficits in mouse model of neurofiromtosis type 1. Nture 415, (22). nture NEUROSCIENCE VOLUME 17 NUMBER 12 DECEMBER

9 npg 214 Nture Americ, Inc. All rights reserved. ONLINE METHODS Mice. mice were ckcrossed to 129S6/SvEv, nd Ptpn11 N38D/+ mice were ckcrossed to C57Bl/6J mice t lest six times efore experiments. Three- to six-month-old mle nd femle mice were used. For AAV experiments, 3- to 4-month-old mle C57Bl/6J mice (Jckson Lortory) were used. Mice were rndomly ssigned to tretment nd experimentl condition. All experiments used littermtes s controls nd were crried nd nlyzed with the experimenters linded to genotype nd tretment. Animls were group housed (2 4) on 12-h light-drk cycle in vivrium t UCLA nd CAU. All studies were pproved y the Animl Reserch Committee t UCLA nd CAU. Drugs. (Tocris) ws dissolved in DMSO (16 mg/ml) nd ws injected intrperitonelly (i.p.) once dily, 3 min efore the wter mze experiment t dose of 32 mg per kilogrm of ody weight. The volume of single injection ws <8 µl. sttin (Mevinolin, Sigm) ws prepred s previously descried 22. Briefly, lovsttin ws dissolved in ethnol (finl concentrtion of 8%) nd 1N NOH ws dded to convert mevinolin to the sodium slt. The ph of the finl solution (4 mg/ml) ws djusted to 7.5 with HCl. The vehicle solution ws prepred with the sme procedure. sttin ws dministered dily (sucutneous injection, 1 mg per kilogrm of ody weight) for 3 d efore the first trining dy of the wter mze nd 6 h efore trining every dy therefter. AAV. The coding sequence of humn PTPN11 with or without the D61G muttion ws sucloned into the HindIII NsiI site of the AAV expression vector psoff. The resultnt vector expresses mutnt PTPN11 under the synthetic CBA promoter (CMV enhncer nd chicken β-ctin promoter). Recominnt virus (raav5) ws purified s previously descried 34. Briefly, n iodixnol grdient purifiction ws performed followed y n ion exchnge chromtogrphy step which results in 99% pure vector preprtion s judged y silver stined-sds crylmide gel frctiontion. After the chromtogrphy, the uffer ws exchnged nd the virus ws concentrted in Ringer s solution using Biomx 1 K concentrtor (Millipore). Vector titers were determined y Rel Time PCR. Typicl titers were genome copies/ml. raav5- expressing only ws used s control. Virus ws infused into two sites per hemisphere (1 µl per injection, AP = 2.5, Lt = ±2, DV = 1.7; AP = 1.8, Lt = ±1, DV = 1.6) over 5 min through 3-guge Hmilton microsyringe. Viruses (crrying, PTPN11 or ) were rndomly ssigned for infusion. After completion of infusion, the syringe ws left in plce for n dditionl 5 min. All the experiments were done 3 weeks fter the infusion. Behvior. Behviorl experiments were performed during the light cycle. In the hidden-pltform version of Morris wter mze, mice were trined with two locks of two trils (inter-tril intervl (ITI) = 1 min) spced ~45 min prt. In ech trining tril, mice were relesed from different strting position nd then were llowed to serch for the escpe pltform for 6 s. The pltform ws sumerged 1 cm under the surfce of the wter. Once mouse found the pltform, it ws left there for 15 s. If mouse did not find the pltform within 6 s, it ws guided to the pltform nd remined on the pltform for 15 s efore eing removed from the pool. Mice were trined for 5 7 consecutive dys. Memory ws ssessed in proe trils tht were given fter completion of trining. During the proe trils, the pltform ws removed nd the mice were llowed to serch for it for 6 s. One mouse ws excluded from further nlysis ecuse of floting (no voluntry movement for more thn 1 s in more thn two trils). The sme group of mice ws tested in the visile-pltform version of Morris wter mze. Dt were cquired nd nlyzed using WterMze softwre (Actimetrics). The oject-plce recognition tsk included trining nd test session. Before trining, mice were hndled 5 min per dy for 4 d nd then hituted in squre ox (27.5 cm 27.5 cm 25 cm) for 15 min for nother 2 d. One side of the experimentl ox included prominent cue. During the 1-min trining session, mice were plced in the ox, exposed to two identicl ojects nd llowed to explore these ojects. During the test session (24 h fter trining), mice were plced ck into the experimentl ox with the sme two ojects for 5 min: one oject (old loction) styed in the sme loction s during trining, while the other (new loction) ws moved to new loction. For the rescue experiment, (32 mg per kilogrm of ody weight, i.p.) ws injected 3 min efore the trining session. The ojects chnged during the test sessions were rndomly counterlnced etween mice. Experiments were videotped nd the explortion times were mnully nlyzed. Electrophysiology. For extrcellulr recordings of field excittory postsynptic potentils (fepsp), sgittl slices (4 µm) were prepred with virtome (VT1S, Leic) in ice-cold rtificil cererospinl fluid (ACSF). Slices recovered t room temperture for t lest 9 min efore recording in ACSF sturted with 95% O 2 nd 5% CO 2 contining the following (in mm): 12 NCl, 3.5 KCl, 2.5 CCl 2, 1.3 MgSO 4, 1.25 NH 2 PO 4, 2 NHCO 3 nd 1 d-glucose. Recording ws performed in sumerged chmer perfused with ACSF (32 C). fepsps were recorded with pltinum-iridium electrodes plced in the CA1 strtum rditum. Bipolr pltinum stimulting electrodes were plced in Schffer collterls. Bseline responses were mesured with stimultion (.17 Hz,.1 ms pulse durtion) t n intensity (typiclly 2 3 µa) tht evoked response tht ws pproximtely one-third of the mximum evoked response. LTP ws induced with TBS (2 or 5 ursts, ech urst consisting of four pulses t 1 Hz with 2 ms inter-urst intervl). Initil fepsp slopes were mesured nd normlized to the verge of seline (with Clmpfit 1.2). Whole-cell voltge clmp recordings were done with n Axoptch 2B mplifier (Axon Instrument) s previously descried 31,32. Coronl slices (35 µm) were prepred in ice-cold slice cutting solution contining the following (in mm), 14 2-hydroxy-N,N,N-trimethylethnminium chloride (choline chloride), 3 sodium pyruvte, 2.5 KCl, 1 CCl 2, 7 MgSO 4, 26 NHCO 3, 3 d-glucose, 1 kynurenic cid, 1.3 sodium scorte. Ptch electrodes (3 6 MΩ when filled) were filled with solution contining the following (in mm): 14 cesiummethnesulfonte, 7 NCl, 1 HEPES,.2 EGTA, 4 Mg-ATP,.3 N-GTP, 5 QX-314. For mepsc recordings, voltge clmp recordings were performed t 6 mv in the presence of 1 µm picrotoxin nd 1 µm TTX. mipscs were mesured t +1 mv in the presence of 1 mm kynurenic cid nd 1 µm TTX. Only recordings during which series resistnce chnged less thn 2% throughout the experiment were nlyzed. mpscs were nlyzed with n in-house nlysis softwre (EVAN) 35. For AMPA/NMDA currents rtio experiments, recordings were performed in ACSF contining 1 µm picrotoxin. Pyrmidl neurons in CA1 were voltge-clmped t 65 mv, nd AMPA-medited EPSCs were evoked y stimulting with ipolr pltinum stimulting electrode t.1 Hz. After recording 15 responses, the holding potentil ws mnully chnged to +4 mv to record NMDA receptor medited EPSCs. The AMPA/NMDA rtio ws clculted y dividing the men vlue of 15 AMPA-medited EPSC pek mplitudes y the men vlue of 15 NMDA receptor medited EPSC mplitudes mesured t 5 ms fter the onset of stimultion (Clmpfit 1.2). Western lotting nd immunohistochemistry. Dissected hippocmpi were homogenized in protein lysis uffer (1 mm Tris-HCl (ph 6.8), 1.6% SDS) contining protese nd phosphtse inhiitor cocktils (Sigm). Superntnts were collected fter centrifugtion nd the protein concentrtion ws determined using BCA ssy kit (Thermo). Equl mounts of proteins (5 µg) were seprted y electrophoresis on 4 12% SDS-PAGE gel (Invitrogen) nd then trnsferred to nitrocellulose memrnes. After locking with 5% BSA in TBS-T (Tris-uffer sline contining.1% Tween-2) for 1 h t room temperture, memrnes were hyridized with primry ntiody overnight t 4 C. After wshing with TBS-T, memrnes were incuted with secondry ntiody in 5% nonft milk nd TBS-T for 1 h t room temperture. Signls were visulized y ECL (Thermo), nd exposure time ws djusted so tht the signls mesured were in liner rnge. After detecting p-erk, the memrnes were stripped nd re-proed with totl Erk ntiody. The totl Erk levels were used to normlize ech smple. The following primry ntiodies were used: nti-p-erk (#911S, Cell Signling, 1:6,), nti-totl Erk (#912S, Cell Signling, 1:5,) nd nti-shp2 (sc-28, Snt Cruz, 1:3,). For immunohistochemistry of SHP2, raav5- or raav5- injected mice were perfused with ice-cold 4% PFA nd the rins were removed nd post-fixed in 4% PFA overnight t 4 C. Coronl rin sections (3 µm thick) were mounted onto slide glsses nd were treted with.3% H 2 O 2 in methnol for 3 min to quench endogenous peroxidse ctivity. After locking with 5% norml got serum in TBS-T (.1% Triton X-1), sections were incuted with nti- SHP2 ntiody (1:1; Sc-28, Snt Cruz Biotechnology) for 48 h t 4 C. A iotinylted nti-rit ntiody (1:5, 1 h t room temperture; Vector Lortories) ws used s secondry, which ws followed y vidin-iotin-peroxidse nture NEUROSCIENCE doi:1.138/nn.3863