In Vivo AAV1 Transduction With hrheb(s16h) Protects Hippocampal Neurons by BDNF Production

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1 originl rticle In Vivo AAV1 Trnsduction With hrhe(s16h) Protects Hippocmpl Neurons y BDNF Production Min-Te Jeon 1,2, Jin Hn Nm 3,4, Won-Ho Shin 5, Eunju Leem 1,2, Kyoung Hoon Jeong 1,2, Un Ju Jung 6, Young-Seuk Be 1,2, Young-Ho Jin 7, Nikoli Kholodilov 8, Roert E Burke 8,9, Seok-Geun Lee 10,11, Byung Kwn Jin 3,4 nd Sng Ryong Kim 1,2,12,13 1 School of Life Sciences, Kyungpook Ntionl University, Degu, Kore; 2 BK21 plus KNU Cretive BioReserch Group, Kyungpook Ntionl University, Degu, Kore; 3 Deprtment of Biochemistry nd Moleculr Biology, School of Medicine, Kyung Hee University, Seoul, Kore; 4 Neurodegenertion Control Reserch Center, School of Medicine, Kyung Hee University, Seoul, Kore; 5 Kore Institute of Toxicology, Kore Reserch Institute of Chemicl Technology, Yuseong, Dejeon, Kore; 6 Center for Food nd Nutritionl Genomics Reserch, Kyungpook Ntionl University, Degu, Kore; 7 Deprtment of Physiology, School of Medicine, Kyung Hee University, Seoul, Kore; 8 Deprtment of Neurology, Columi University, New York, New York, USA; 9 Deprtment of Pthology nd Cell Biology, Columi University, New York, New York, USA; 10 Cncer Preventive Mteril Development Reserch Center, Deprtment of Science, College of Koren Medicine, Kyung Hee University, Seoul, Kore; 11 Koren Medicine Clinicl Tril Center, Kyung Hee University Koren Medicine Hospitl, Seoul, Kore; 12 Institute of Life Science nd Biotechnology, Kyungpook Ntionl University, Degu, Kore; 13 Brin Science nd Engineering Institute, Kyungpook Ntionl University, Degu, Kore Recent evidence hs shown tht Rs homolog enriched in rin (Rhe) is dysregulted in Alzheimer s disese (AD) rins. However, it is still uncler whether Rhe ctivtion contriutes to the survivl nd protection of hippocmpl neurons in the dult rin. To ssess the effects of ctive Rhe in hippocmpl neurons in vivo, we trnsfected neurons in the cornu mmonis 1 (CA1) region in norml dult rts with n deno-ssocited virus contining the constitutively ctive humn Rhe (hrhe(s16h)) nd evluted the effects on thromininduced neurotoxicity. Trnsduction with hrhe(s16h) significntly induced neurotrophic effects in hippocmpl neurons through ctivtion of mmmlin trget of rpmycin complex 1 (mtorc1) without side effects such s long-term potentition impirment nd seizures from the ltertion of cytorchitecture, nd the expression of hrhe(s16h) prevented thromininduced neurodegenertion in vivo, n effect tht ws diminished y tretment with specific neutrlizing ntiodies ginst rin-derived neurotrophic fctor (BDNF). In ddition, our results showed tht the sl mtorc1 ctivity might e insufficient to medite the level of BDNF expression, ut hrhe(s16h)-ctivted mtorc1 stimulted BDNF production in hippocmpl neurons. These results suggest tht virl vector trnsduction with hrhe(s16h) my hve therpeutic vlue in the tretment of neurodegenertive diseses such s AD. Received 29 April 2014; ccepted 6 Decemer 2014; dvnce online puliction 13 Jnury doi: /mt INTRODUCTION Alzheimer s disese (AD) is the most common neurodegenertive disorder nd is chrcterized y impired cognitive function, prticulrly for memory. 1 There is no cure for the disese, which worsens s it progresses, nd eventully cuses deth. In contrst to known pthologicl hllmrks including neuronl degenertion, extrcellulr neuritic plques, nd intrcellulr neurofirillry tngles, the pthogenetic mechnisms underlying neurodegenertion remin lrgely unknown, nd tretments tht re le to interfere with the progression of the disese re limited. 2 However, despite the lck of complete understnding of the etiology of AD for guiding the development of knowledge-sed trgeted therpeutics, which includes incresed levels of mmmlin trget of rpmycin complex 1 (mtorc1) in the rins of ptients with AD, 2 4 numer of studies suggest tht the control of mtorc1 signling pthwy could e eneficil strtegy for the protection nd functionl mintennce of hippocmpl system in the rin. 5 8 Rs homolog enriched in rin (Rhe) is memer of the Rs fmily of smll GTP-inding proteins. It medites the ctivtion of mtorc1, which enhnces the ctivity of intrcellulr cell survivl pthwys The serine t position 16 of Rhe hs sensitivity to tuerous sclerosis complex GTPse ctivtion, nd Rhe(S16H), muttion of the serine to histidine, results in persistence of the GTP-ound Rhe s n ctivted stte owing to the resistnce to tuerous sclerosis complex ctivtion In nigrl dopminergic neurons in vivo, deno-ssocited virus 1 (AAV1) trnsduction with gene encoding humn Rhe(S16H) (hrhe(s16h)) induces trophic effects resulting in the protection of the dopminergic system in neurotoxin model of Prkinson s disese. 12,13 Correspondence: Sng Ryong Kim, School of Life Sciences, Kyungpook Ntionl University, Degu , Kore. E-mil: srk75@knu.c.kr or Byung Kwn Jin, Deprtment of Biochemistry nd Moleculr Biology, School of Medicine, Kyung Hee University, Seoul , Kore. E-mil: kjin@khu.c.kr Moleculr Therpy vol. 23 no. 3, mr

2 In the hippocmpus, Rhe mrna is rpidly nd trnsiently induced in hippocmpl grnule cells y seizures nd y N-methyl-sprtte-dependent synptic ctivity in longterm potentition (LTP) prdigm. 15 However, recent results hve shown tht Rhe is dysregulted in the rins of ptients with AD, nd its overexpression promotes the degrdtion of β-site myloid precursor protein-cleving enzyme 1 (BACE1) in model of AD. 16 These findings suggest tht Rhe my e n importnt regultor of the survivl of hippocmpl neurons in the dult rin. Although the effects of Rhe on the level of BACE1 re mtorc1 independent, 16 it is lrgely unknown whether the ctivtion of Rhe/mTORC1 signling pthwys induces neurotrophic effects in the hippocmpus nd protects hippocmpl neurons ginst neurotoxicity. In this study, we therefore investigted the effects of mtorc1 ctivtion through the trnsduction of hippocmpl neurons with hrhe(s16h) in the norml rt rin nd the neuroprotective mechnisms of hrhe(s16h) ginst thromin-induced neurodegenertion in the hippocmpus. RESULTS Neurotrophic effects of hrhe(s16h) in hippocmpl neurons in norml dult rt rins AAV-hRhe(S16H) or AAV-green fluorescent protein () s control vector ws unilterlly injected into the hippocmpus y trgeting the cornu mmonis 1 (CA1) region of norml dult rts. Four weeks lter, the rins were removed, nd sections were processed to determine the trnsduction of hippocmpl neurons y the virl injection. The trnsduction of hippocmpl neurons with nd hrhe(s16h) ws demonstrted y the immedite oservtion of expression nd immunoperoxidse stining for the FLAG epitope (Figure 1), respectively, nd y triple immunofluorescence leling for 4,6-dimidino-2-phenylindole,, nd neuronl nuclei (NeuN) or 4,6-dimidino-2-phenylindole, FLAG, nd NeuN (Figure 1), respectively. Immunofluorescence leling of microgli (OX-42 nd ionized clcium-inding dpter molecule 1 (I1)-immunopositive cells) nd strocytes (glil firillry cidic protein (GFAP)-immunopositive cells) indicted no trnsduction in ech cell type, indicting the specific trnsduction of AAV-hRhe(S16H) nd AAV- in the hippocmpl neurons (Supplementry Figure S1). We next exmined whether the hrhe(s16h) trnsduction of hippocmpl neurons induced n increse in phosphoryltion of the mtorc1 sustrtes 4E-BP1 nd p70s6k, which indictes mtorc1 ctivtion, nd ssessed the neurotrophic effects in hippocmpl neurons. Similr to our recent results on mtorc1 ctivtion y hrhe(s16h) in dult dopminergic neurons, 13 the levels of phospho-4e-bp1 (p-4e-bp1) nd phospho-p70s6k (p-p70s6k) were significntly incresed in rts trnsduced with hrhe(s16h) compred to intct controls nd -treted rts, indicting mtorc1 ctivtion in hippocmpl neurons, s demonstrted y immunohistochemicl (Figure 2) nd western lot nlysis (P < 0.01 versus controls; Figure 2,c). nd FLAG were strongly detected in smples sujected to ech virl injection, indicting the successful trnsduction of AAV-hRhe(S16H) nd AAV-, respectively (Figure 2), nd hrhe(s16h) expression incresed the hippocmpl levels of totl choline with modest hrhe(s16h) DAPI /FLAG NeuN Merge Figure 1 Trnsduction of hippocmpl neurons with AAV- nd AAV-hRhe(S16H) in norml dult SD rts. () Animls received unilterl injection of AAV- or AAV-hRhe(S16) in the hippocmpus nd were scrificed 4 weeks lter. AAV-hRhe(S16H)-injected rin sections (30 μm) were processed for immunostining with ntiodies ginst FLAG. The expression of nd FLAG (rown rection product) is oserved in the hippocmpus for ech virl injection, ut no expression of FLAG is seen in the noninjected control side (). Br = 100 μm. () Immunofluorescence triple leling for DAPI (lue), (green), nd NeuN (red), or DAPI, FLAG (green), nd NeuN shows tht trnsgene expression is identifile within neurons in the CA1 of hippocmpus. Br = 20μm. All of the pictures show the representtive coronl sections following ech immunostining (n = 3, ech group). ltertion in level of cetylcholine, which re importnt for cognitive function, lerning, nd memory performnce (P = nd P = 0.117, respectively, versus controls; Supplementry Figure S2). In ddition to the ctivtion of mtorc1 nd the increse in totl choline, hrhe(s16h) induced morphologicl chnges to hippocmpl neurons, s demonstrted y Nissl stining (Figure 3) nd NeuN immunostining (Figure 3c), indicting the incresed re of neurons with hrhe(s16h) expression compred to the intct controls nd -expressed controls (P < 0.01 versus controls; Figure 3). The cytorchitecturl normlities of hippocmpl neurons could e involved in neuronl circuitry impirment 20 or norml ehviorl chnges. 21 To scertin if there were side effects such s impired LTP nd norml ehviors from the morphologicl chnges in the hippocmpl neurons, we dditionlly investigted the effects of hrhe(s16h) on chnges in LTP in the hippocmpus nd on norml ehvior, such s seizures. Our results showed tht the hrhe(s16h)-induced morphologicl chnges of hippocmpl neurons did not ffect sl LTP in the hippocmpus (Figure 3e) nd did not cuse ehviorl disorders, such s seizures, compred to kinic cid-induced ehviorl normlities (Supplementry Figure S3), suggesting tht the hrhe(s16h) trnsduction of hippocmpl neurons induces cellulr morphologic chnges without side effects such s neuronl circuitry impirment or seizures in the hippocmpus. Similr to the effects in the sustnti nigr of dult vol. 23 no. 3 mr. 2015

3 hrhe (S16H) p-4e-bp1 (T37/46) 4E-BP1 p-p70s6k p70s6k FLAG β-action hrhe(s16h) c Opticl density (ritry units) p-4e-bp1 (T37/46) hrhe(s16h) 4E-BP1 p-p70s6k p70s6k Figure 2 hrhe(s16h) expression ctivtes mtorc1 in the hippocmpus. () Brin sections were stined with nti-phospho-4e-bp1, mtorc1 sustrte, t 4 weeks postinjection of virl vectors. Immunoperoxidse stining for p-4e-bp1 (with thionin counterstin) shows tht rown rection products re clerly oserved in the neurons of the hrhe(s16h)-treted group, compred to modest level in the vehicle-treted group. Br = 500 μm. Insets show mgnified photomicrogrphs of the re in the CA1 lyer. An exmple of neuronl p-4e-bp1 stining (white rrows) is shown in the inset. All pictures show the representtive coronl section of ech group (n = 3, ech group). () Western lot nlysis of p-4e-bp1, 4E-BP1, p-p70s6k, nd p70s6k expression t 4 weeks fter intrhippocmpl injection of AAV- nd AAV-hRhe(S16H). Successful trnsduction of the hippocmpus ws confirmed in ech cse y western lot nlysis of nd FLAG expressions. (c) The histogrm results show the results of quntittive nlysis sed on the density of the p-4e-bp1, 4E-BP1, p-p70s6k, nd p70s6k nds normlized with the β-ctin nd for ech smple. All vlues represent the men ± SEM of four pooled smples for ech group. P < 0.01, significntly different from contrlterl control side () nd AAV- (one-wy nlysis of vrince nd Student Newmn Keuls nlysis). mice rins, 13 the numer of rt hippocmpl neurons ws not influenced y the virl injection (Figure 3d). Induction of rin-derived neurotrophic fctor y hrhe(s16h) in dult hippocmpl neurons in vivo A numer of studies hve shown reduction in rin-derived neurotrophic fctor (BDNF) expression in the rins of ptients with AD Moreover, BDNF delivery hs neuroprotective effects in niml models of AD, 25,26 suggesting tht the sustined support of BDNF cn e useful for the protection of hippocmpl neurons in the dult rin. The cellulr effects of BDNF cn e medited y the Akt/mTOR signling pthwy in neurons. 27,28 However, it is lrgely unknown whether the increse in mtorc1 ctivity y specific gene delivery cn produce BDNF in hippocmpl neurons in the dult rin, suggesting the role of mtorc1 s n ctivtor of BDNF production, in ddition to its role s downstrem molecule tht is medited y BDNF. We therefore exmined whether the continul stimultion of mtorc1 y trnsduction with hrhe(s16h) induced BDNF expression in hippocmpl neurons in vivo. As demonstrted y the immunohistochemicl nlysis conducted 4 weeks postinjection, hrhe(s16h) induced n increse in the hippocmpl expression of BDNF ut not in -expressed hippocmpus compred to the intct controls (Figure 4), nd the incresed BDNF expression ws pprently oserved in NeuN-positive neurons (Figure 4), indicting BDNF induction y hrhe(s16h) in hippocmpl neurons. Consistent with the BDNF stining, western lot nlysis showed tht hrhe(s16h)-trnsduced hippocmpl side hd significntly incresed levels of BDNF compred to the contrlterl controls (P = 0.002; Figure 4c,d). To scertin whether the induction of BDNF y hrhe(s16h) ws mtorc1 dependent, we further exmined the effects of rpmycin, which is specific inhiitor of mtorc1, 21 on the levels of BDNF expression nd mtorc1 ctivity. Rts received n intrperitonel injection of rpmycin (5 mg/kg) 3 weeks fter the virl injection, nd the tretment ws continued until 4 weeks postinjection. Our results showed tht tretment with 5 mg/kg of rpmycin significntly ttenuted the expression of p-4e-bp1 nd p-p70s6k in the hippocmpus compred to tht in intct controls (Supplementry Figure S4,c), ut its tretment did not lter the sl levels of BDNF in the hippocmpus (P = versus controls; Figure 4c,d). However, it significntly ttenuted the hrhe(s16h)-induced increse in BDNF expression (P < versus hrhe(s16h) lone; Figure 4c,d). To clrify the involvement of mtorc1 in the sl levels of BDNF, we further investigted whether tretment with 10 mg/ kg of rpmycin ttenuted the sl levels of BDNF in the hippocmpus. Although 10 mg/kg of rpmycin lso showed significnt reduction in mtorc1 ctivity compred to the intct controls, it Moleculr Therpy vol. 23 no. 3 mr

4 c hrhe(s16h) hrhe(s16h) EXP d Fold increse (neuronl re % of con) Hippocmpl CA1 neurons (% of con) hrhe (WT) hrhe (S16H) 0 hrhe (WT) hrhe (S16H) e Control hrhe(s16h) 0.5 mv 2 ms fepsp slope (%) Control hrhe(s16h) Time (minutes) Figure 3 hrhe(s16h) induces hypertrophic effect without LTP impirment from the cytorchitecturl chnges in the hippocmpus. () Morphologic nlysis of hippocmpl neurons t 4 weeks fter intrhippocmpl injection of AAV-hRhe(S16H). The upper pnel shows representtive coronl section of the hippocmpus following Nissl stining y cresyl violet. The experimentl side (EXP) injected with AAV-hRhe(S16H) shows n increse in the re of Nissl-positive neurons, compred to the contrlterl control side (), s shown in representtive microgrphs t higher power in the lower pnels. All pictures show the representtive coronl section of ech group (n = 5, ech group). Br = 500 μm (upper pnel) nd 20 μm (lower pnel). () The re of Nissl-positive neurons in the CA1 lyer. The re of neurons ws expressed quntittively s percentge reltive to the contrlterl control. P < 0.01, significntly different from nd AAV- (one-wy nlysis of vrince nd Student Newmn Keuls nlysis; n = 5, ech group). WT, wild-type. (c) NeuN-positive neurons lso show morphologic chnges in the hippocmpus t 4 weeks fter injection of hrhe(s16h). (d) The numer of the NeuN-positive neurons in the CA1 lyer injected with virl vectors. Note tht there is no difference etween groups (one-wy nlysis of vrince nd Student Newmn Keuls nlysis; n = 5, ech group). (e) Effect of hrhe(s16h) on the induction of LTP in the hippocmpus. Hippocmpl slices were prepred from the rts 3 weeks (n = 6) fter injection of hrhe(s16h), nd noninjected slices were used s controls (n = 6). The slices were perfused with rtificil cererospinl fluid (ACSF) for 60 minutes. For the induction of LTP, the slices were tetnized with 100 Hz for 1 second stimultion t time 0 minute. Note tht there is no difference etween the groups. Ech point nd line represents the men ± SEM. Representtive field excittory postsynptic potentil (fepsp) recordings t time 5 nd 30 minutes re shown s insets (upper side). did not show ny significnt reductions in sl BDNF expression (Supplementry Figure S4,d). Tken together, these results suggest tht hrhe(s16h)-ctivted mtorc1 my induce the production of BDNF in the hippocmpl neurons, even though the sl levels of BDNF re not ffected y mtorc1. Neuroprotective effects of hrhe(s16h) ginst thromin-induced neuronl cell deth in the CA1 of rt hippocmpus Incresed levels of thromin, which result in the deth of hippocmpl neurons nd AD-like cognitive impirment, hve een vol. 23 no. 3 mr. 2015

5 hrhe(s16h) NeuN BDNF Merge hrhe(s16h) c d BDNF β-ction Rpmycin hrhe(s16h) Opticl density (ritrry units) P = # Rpmycin hrhe(s16h) Figure 4 hrhe(s16h) increses BDNF expression in hippocmpl neurons. () Immunoperoxidse stining for BDNF ws performed t 4 weeks postinjection of AAV-hRhe(S16H). Both the contrlterl control side () nd the AAV--injected side () show similr levels of BDNF in the hippocmpus. However, the AAV-hRhe(S16H)-injected side (hrhe(s16h)) shows n increse in the level of BDNF in the hippocmpus. Br = 500 μm. () Immunofluorescence doule stining for NeuN (green) nd BDNF (red) shows tht the hrhe(s16h)-incresed expression of BDNF is identifile within hippocmpl neurons. Br = 100 μm. Insets re higher mgnifictions of ech CA1 lyer. All pictures in nd show representtive coronl sections following ech immunostining (n = 3, ech group). (c) Western lot nlysis of BDNF in the hippocmpus. Rts were intrperitonelly injected with rpmycin (5 mg/kg), strting 3 weeks fter AAV-hRhe(S16H) injection nd continued dily injection until 4 weeks postinjection, nd then they were scrificed for western lot nlysis. The results show tht tretment with 5 mg/kg rpmycin lone cuses no ltertion to the sic level of BDNF compred to the intct control (first nd). However, its tretment ttenuted the level of hrhe(s16h)-incresed BDNF. (d) All vlues of the opticl density of ech nd represent the men ± SEM of six pooled smples in ech group. P = nd # P < 0.001, significntly different from nd hrhe(s16h) lone, respectively (one-wy nlysis of vrince nd Student Newmn Keuls nlysis). oserved in AD rins This suggests tht thromin my e n importnt endogenous pthogen for AD. Given the pronounced trophic effects of hrhe(s16h) in norml rts, we next investigted whether hrhe(s16h) hd neuroprotective effects in thromin-treted rt model of AD. 29 Thromin (20 U) or phosphte-uffered sline (PBS), which ws used s control (dt not shown), ws unilterlly injected into the hippocmpus. Seven dys lter, the rins were removed, nd the sections were processed for nti-neun immunostining (Figure 5 nd Supplementry Figure S5). As previously reported, 29 there ws significnt loss of NeuN-positive neurons in the thromin-treted CA1, compred to the control CA1 (P < versus controls; Figure 5, nd Supplementry Figure S5). However, the AAV trnsduction of hippocmpl neurons with hrhe(s16h) 3 weeks efore the intrhippocmpl injection of thromin provided significnt neuroprotection s demonstrted y the immunohistochemicl nlysis (Figure 5, nd Supplementry Figure S5). When quntified nd expressed s percentge of the CA1 neurons in the counting re of the ipsilterl hippocmpus reltive to the contrlterl control, only 42% of the hippocmpl neurons were preserved in the hippocmpl CA1 region of the thromin-treted rts, wheres 95% of the hippocmpl neurons were preserved in the presence of hrhe(s16h) (P < versus thromin; Figure 5). Moreover, the levels of totl choline nd cetylcholine incresed y hrhe(s16h) trnsduction were preserved in the thromin-treted hippocmpus (Supplementry Figure S6). Although thromin lone shows no reduction in oth levels Moleculr Therpy vol. 23 no. 3 mr

6 Thromin neurotoxicity in the hippocmpus. As demonstrted y western lot nlysis (Supplementry Figure S7,), our results unexpectedly showed tht thromin tretment significntly incresed the levels of p-4e-bp1 compred to the intct controls. However, immunohistochemicl stining for p-4e-bp1 showed tht its incresed expression y thromin ws loclized within nonneuronl cells, nd, on the contrry, its expression ws decresed in hippocmpl neurons (Supplementry Figure S7c,d). These results suggest tht neuronl mtorc1 ctivity medited y hrhe(s16h) my e importnt for the neuronl survivl in the thromin-treted hippocmpus. Hippocmpl CA1 neurons (% of con) thromin hrhe(s16h) thromin Thromin Figure 5 hrhe(s16h) protects hippocmpl neurons from thromin-induced neurotoxicity in vivo. () Rts received n intrhippocmpl injection of thromin (20 U) t 3 weeks postinjection of virl vectors, nd NeuN immunostining ws ssessed t 1 week following thromin tretment. Br = 500 μm. The right pnels show higher mgnifictions of ech CA1 lyer. Br = 20 μm. All pictures show the representtive coronl section of ech group (n = 5, ech group). () The histogrm results show the numer of hippocmpl neurons in the trget re of the CA1 lyer, which is expressed quntittively s percentge compred to the contrlterl control. The results show tht hrhe(s16h) hs protective effects ginst thromin-induced neurotoxicity. P < nd # P < 0.001, significntly different compred to the contrlterl control () nd thromin lone, respectively (one-wy nlysis of vrince nd Student Newmn Keuls nlysis; n = 5, ech group). of totl choline nd cetylcholine compred with intct controls (Supplementry Figure S6), these results suggest tht hrhe(s16h) is cple of inducing neuroprotection ginst the thromin-induced deth of hippocmpl neurons. However, it ws still uncler whether the incresed levels of thromin sustntilly reduced the expression of p-4e-bp1 in the hippocmpl neurons tht my e involved in the neuroprotection from hrhe(s16h) trnsduction ginst thromin-induced hrhe (S16H) thromin thromin # BDNF contriuted to the neuroprotection induced y hrhe(s16h) in the thromin-treted hippocmpus To evlute whether the induction of BDNF correlted with hrhe(s16h)-induced neuroprotection in the thromintreted rt model of AD, rts received unilterl injection of neutrlizing ntiodies ginst BDNF (100, 200, or 400 ng) with thromin (20 U) into the hippocmpl CA1 region 3 weeks fter hrhe(s16h) tretment, nd the rins were nlyzed y immunohistochemicl stining 1 week fter injection of neutrlizing ntiodies (Figure 6,). The results showed tht the protection y hrhe(s16h) ginst thromin-induced neurotoxicity tht ws descried in Figure 5 ws ttenuted y tretment with neutrlizing ntiodies ginst BDNF in dose-dependent mnner (Figure 6). When quntified nd expressed s percentge of CA1 neurons in the counting re of the ipsilterl hippocmpus compred to the contrlterl controls, the dministrtion of 100 ng BDNF neutrlizing ntiodies preserved the numer of NeuN-positive neurons y 83%, indicting modest inhiitory effect on the hrhe(s16h)-induced protection of hippocmpl neurons (P = versus hrhe(s16h)thromin). However, tretment with 200 nd 400 ng of neutrlizing ntiodies significntly ttenuted the preservtion of NeuNpositive neurons y 67 nd 61%, respectively (P < 0.01 versus hrhe(s16h)thromin). Rts treted with neutrlizing ntiodies lone in the sence of hrhe(s16h) showed no ltertions in the numer of NeuN-positive neurons (Supplementry Figure S8). To scertin the effects of BDNF neutrliztion on mtorc1 ctivity, we further exmined the chnges in 4E-BP1, p70s6k, p-4e-bp1, nd p-p70s6k expression induced y BDNF neutrlizing ntiodies in the sence or presence of hrhe(s16h). Rts received unilterl injection of 300 ng neutrlizing ntiodies ginst BDNF into the hippocmpl CA1 region 3 weeks fter hrhe(s16h), nd the rins were nlyzed y western lotting 2 dys fter the injection of neutrlizing ntiodies. Although the tretment with neutrlizing ntiodies resulted in no ltertions in sic mtorc1 ctivity in the hippocmpus, it significntly ttenuted the levels of p-4e-bp1 nd p-p70s6k tht were incresed y hrhe(s16h) compred to hrhe(s16h) lone (Supplementry Figure S9), suggesting tht BDNF neutrliztion inhiited the ctivtion of mtorc1 induced y hrhe(s16h) trnsduction in the hippocmpus. Thus, ll of these results show tht the ctivtion of the hrhe(s16h)/ mtorc1 signling pthwy cn protect hippocmpl neurons through the production of BDNF vol. 23 no. 3 mr. 2015

7 Hippocmpl CA1 neurons (% of con) Thromin hrhe(s16h) Thromin BDNF NA 100 BDNF NA 200 BDNF NA 400 hrhe(s16h) thromin P = hrhe(s16h)thromin BDNF NA 400 ng Figure 6 The induction of BDNF y hrhe(s16h) contriutes to the protection of hippocmpl neurons. () Rts received n intrhippocmpl injection of mixture of BDNF neutrlizing ntiodies (100, 200 or 400 ng) nd thromin (20 U) t 3 weeks fter AAV-hRhe(S16H) injection. Brins were removed nd processed for NeuN immunostining t 1 week following injection of the mixture. As descried in Figure 5, hrhe(s16h) protects hippocmpl neurons from thromin-induced neurotoxicity. However, tretment with BDNF neutrlizing ntiodies ttenutes hrhe(s16h)-induced neuroprotection. All pictures show the representtive coronl section of ech group (n = 5, ech group). Brs = 500 nd 20 μm, respectively. () The numer of NeuN-positive hippocmpl neurons in the trget re of the CA1 lyer ws expressed quntittively s percentge compred to the contrlterl control. The results show tht hrhe(s16h)-induced BDNF contriutes to neuroprotection in the thromin-treted hippocmpus. P < 0.001, # P < 0.001, P < 0.05, nd ## P < 0.01, significntly different compred with contrlterl control side (), thromin lone,, nd hrhe(s16h)thromin, respectively (one-wy nlysis of vrince nd Student Newmn Keulsn nlysis; n = 5, ech group). DISCUSSION It is controversil whether the ctivtion of mtorc1 hs neuroprotective effects in the hippocmpus of dult rins ecuse there is no evidence for the direct mtorc1-medited protection of hippocmpl neurons, the incresed levels of mtorc1 in AD rins, 3,4 nd the correltion etween mtorc1 ctivtion nd cognitive severity of AD ptients. 2 However, there re numer of studies descriing the puttive role of mtorc1 in protecting hippocmpl neurons nd the functionl mintennce of hippocmpl system. 5 8 In ddition to the previous results, Chen et l. 28 hve # ## ## reported tht myloid-β, which plys centrl role in AD, interrupts the mtor signling pthwy in rt corticl neurons in vitro. Furthermore, kinic cid tretment, which is well-estlished model of hippocmpl neuron deth, induces the ctivtion of Akt/mTOR signling pthwys, which correltes with the control of utophgic stress. 33 Tken together, these results suggest tht incresed levels of mtorc1 in the rins of ptients with AD my trigger potentil negtive feedck loop to inhiit further neurotoxicity, nd the stimultion of the mtorc1 signling pthwy could e n importnt strtegy for the control of neurodegenertion in the hippocmpus. GTP-ound ctive Rhe stimultes the ctivtion of mtorc1, 14 nd constitutively ctive form of Rhe such s hrhe(s16h) pprently ctivtes mtorc1 in dult neurons in vivo. 12,13 Recently, Shhni et l. 16 reported tht Rhe is dysregulted in AD rins, nd Rhe overexpression promotes the degrdtion of BACE1, which is involved in myloid-β genertion, through mtorc1-independent pthwy in model of AD. 16 This indictes tht Rhe my e n importnt regultor of the survivl of hippocmpl neurons in the dult rin. However, it is still uncler whether the induction of ctive Rhe in hippocmpl neurons cn contriute to neuroprotection in vivo nd whether the Rhe-induced effects re mtorc1 dependent or independent. In this study, we first investigted whether AAV1 trnsduction with gene encoding hrhe(s16h) induced trophic effects through mtorc1 ctivtion in the hippocmpl neurons in vivo. 4E-BP1, which is representtive sustrte of mtorc1, hs seven phosphoryltion sites. 34,35 Its phosphoryltion proceeds in sequentil mnner, 34 nd the Thr37/46 sites re directly phosphorylted y the ctivtion of mtorc1. 35 Here, we demonstrted tht hrhe(s16h) trnsduction significntly incresed 4E-BP1 phosphoryltion t Thr37/46 in the hippocmpus of norml dult rts compred to the intct controls nd controls (Figure 2). In ddition to n increse in p-4e-bp1 level, phosphoryltion of p70s6k, which is nother sustrte of mtorc1, ws lso incresed y hrhe(s16h) trnsduction (Figure 2). These results suggest tht the trnsduction of hippocmpl neurons with hrhe(s16h) cn pprently increse mtorc1 ctivity in the hippocmpus in vivo. In norml dult rt rins, the levels of cetylcholine nd totl choline, which re importnt for cognitive function, lerning, nd memory performnce, were incresed in the hrhe(s16h)- expressed hippocmpus, even though cetylcholine level showed modest ltertion with no significnt chnge compred to tht in the intct hippocmpus (Supplementry Figure S2). hrhe(s16h) expression led to increses in the re of neurons in the hippocmpus (Figure 3) due to hypertrophic effect in neurons 13,36 nd in the levels of neuronl BDNF, which is wellrecognized neurotrophic fctor ginst AD 28,37,38 (Figure 4). The cytorchitecturl normlities in hippocmpl neurons could e involved in neuronl circuitry impirments 20 or norml ehviorl chnges. 21 However, our investigtions to clrify these side effects from the chnges in cytorchitecture showed tht the hrhe(s16h) trnsduction of hippocmpl neurons hd no effect on chnges in LTP (Figure 3) nd norml ehviors, such s seizures (Supplementry Figure S3), suggesting tht hrhe(s16h) expression ws not involved in the ltertions in the Moleculr Therpy vol. 23 no. 3 mr

8 synptic structure in the hippocmpus. 39 We therefore conclude tht trnsduction of hippocmpl neurons with hrhe(s16h) cn induce neurotrophic effects without side effects such s neuronl circuitry impirment nd seizures in the hippocmpus of norml dult rts, even though there were cellulr morphologic chnges in the hippocmpl neurons. In ddition to the hrhe(s16h)-induced trophic effects in the hippocmpus of norml rts, we evluted the ility of hrhe(s16h) to protect hippocmpl neurons from the thromin-induced neurotoxicity. 29 Thromin, which is serine protese of the trypsin fmily, is key enzyme of the lood cogultion system nd is generted from prothromin y clevge following prothrominse ctivtion. 31 Its expression is incresed in the rins of ptients with AD. It ccumultes in senile plques, rective microglil cells, nd neurofirillry tngles in AD rins nd microvessels, 31,32 wheres the ctivity of the thromin inhiitor, protese nexin I, is shrply reduced in AD rins. 40 Moreover, numer of studies hve shown tht thromin cn ct s neurotoxin, leding to the deth of hippocmpl neurons nd AD-like cognitive impirment, 29,30,32 suggesting tht ltertions in thromin levels my e n importnt endogenous pthogen for AD. In the current study, our oservtions showed tht thromin tretment decresed the expression of p-4e-bp1 in hippocmpl neurons (Supplementry Figure S7), suggesting tht the mintennce of mtorc1 ctivity in hippocmpl neurons my e crucil for neuronl survivl ginst thromin-induced neurotoxicity. Furthermore, our results showed tht hrhe(s16h) hd roust ility to protect hippocmpl neurons from thromin-induced neurotoxicity (Figure 5) with n increse in the levels of totl choline nd cetylcholine in the hippocmpus (Supplementry Figure S6). BDNF is neurotrophin tht medites neuronl survivl nd differentition. 41 A numer of studies hve shown tht BDNF expression is decresed in AD rins nd tht BDNF delivery hs neuroprotective effects in niml models of AD. 25,26 The cellulr effects of BDNF re initited y its inding to the specific receptor, tropomyosin receptor kinse B, 42 nd BDNF tretment ctivtes the Akt/mTOR signling pthwys in neurons. 27 However, it is lrgely unknown whether the ctivtion of Rhe/ mtorc1 signling pthwys gives hippocmpl neurons the ility to produce BDNF, therey contriuting to neuroprotection in the hippocmpus of dult rins. To scertin whether the ctivtion of mtorc1 y hrhe(s16h) medited the induction of BDNF production, we exmined the effects of rpmycin, which is specific inhiitor of mtorc1 (ref. 21), on the levels of BDNF in the hippocmpus. Although rpmycin did not lter the sic levels of BDNF in the hippocmpus (P = versus norml controls), it significntly inhiited n increse in BDNF in the hrhe(s16h)-treted hippocmpus, suggesting tht the incresed BDNF expression my e mtorc1 dependent (Figure 4). Moreover, our oservtions regrding the effects of neutrlizing ntiodies ginst BDNF on hrhe(s16h)-induced neuroprotection indicted tht BDNF neutrliztion could significntly reduce the hrhe(s16h)-incresed mtorc1 ctivity lthough tretment with neutrlizing ntiodies showed no ltertions in sic mtorc1 ctivity in the hippocmpus of norml rt rins (Supplementry Figure S9), suggesting TrkB(?) GDP Rhe(S16H) mtorc1 BDNF Neuroprotection Rpmycin BDNF NA Figure 7 Schemtic representtion of hrhe(s16h)-induced neuroprotection in the hippocmpus. The serine t position 16 of hrhe hs sensitivity to tuerous sclerosis complex (TSC) GTPse ctivtion, nd hrhe(s16h) muttion of the serine to histidine results in resistnce of hrhe to this ctivtion nd consequently persistence of the GTPound, ctivted stte. 13 The ccumultion of hrhe(s16h) stimultes mtorc1, nd the ctivtion of mtorc1 induces BDNF production in hippocmpl neurons. Moreover, hrhe(s16h)-induced BDNF contriutes to the ctivtion of mtorc1, even though it is uncler whether the induction of BDNF ctivtes its specific receptor, tropomyosin receptor kinse B (TrkB), which is expressed in hippocmpl neurons. 48 Tken together, the synergetic effects of hrhe(s16h) nd hrhe(s16h)- induced BDNF on the ctivtion of mtorc1 my contriute to neuroprotection in the hippocmpus. tht hrhe(s16h)-incresed BDNF, t lest in prt, might e involved in the ctivtion of mtorc1 with direct stimultion y hrhe(s16h) nd tht the induction of BDNF contriuted to the protection of hippocmpl neurons from thromin-induced neurotoxicity (Figure 6). Tken together, these results suggest tht the synergetic effects of hrhe(s16h) nd hrhe(s16h)-induced BDNF on the ctivtion of mtorc1 my contriute to neuroprotection in the thromin-treted hippocmpus (Figure 7). In conclusion, we found tht hrhe(s16h) expression hd roust trophic nd protective effects in dult hippocmpl neurons. Moreover, we demonstrted tht the hrhe(s16h) trnsduction of hippocmpl neurons induced the sustined production of BDNF vi mtorc1-dependent pthwy, contriuting to neuroprotection in the dult rin (Figure 7). Although we cnnot exclude the possiility tht nother mechnism my e involved in the production of BDNF y hrhe(s16h) due to the potentil possiility of n interction etween hrhe(s16h) nd rpmycin, the present oservtions suggest tht mtorc1 ctivtion y specific gene delivery to hippocmpl neurons my e useful strtegy for protecting neurons in the hippocmpus of dult rins. MATERIALS AND METHODS Production of AAV virl vectors. All vectors used for these studies were AAV1 serotype s previously descried. 12,13 A plsmid crrying the hrhe ws purchsed from OriGene Technologies (Rockville, MD). hrhe DNA ws mplified nd modified to incorporte FLAG-encoding sequence t the 3 -end y expnded long-templte PCR (Roche, Indinpolis, IN). Constitutively ctive hrhe (hrhe(s16h)) ws generted y use of the Phusion Site-directed Mutgenesis Kit of New Englnd Biols (Ipswich, MA) in the pgem-t vector (Promeg, Sn Luis Oispo, CA) nd then cloned into n AAV pckging construct tht utilizes the chicken β-ctin promoter nd contins 3 WPRE (pbl). AAVs were produced y GTP TSC1 TSC vol. 23 no. 3 mr. 2015

9 the University of North Crolin Vector Core, nd the genomic titer of hrhe(wt) nd hrhe(s16h) were nd virl genomes/ml, respectively. Enhnced green fluorescent protein (), used s control, ws sucloned into the sme virl ckone, nd virl stocks were produced t titers of virl genomes/ml. Institutionl review of niml protocols. Sprgue Dwley (SD) rts (10-week-old, g) were otined from Dehn Biolink (Eumseong, Kore). All surgicl experiments were performed in ccordnce with pproved niml protocols nd guidelines estlished y the Animl Cre Committee of Kyungpook Ntionl University (no. KNU ). Intrhippocmpl AAV injection. SD rts were nesthetized y intrperitonel injection of chlorl hydrte (360 mg/kg; Sigm, St Louis, MO) nd plced in stereotxic frme (Dvid Kopf Instrument, Tujung, CA). Ech rt received unilterl injection of AAV- s control vector or AAVhRhe using 10 μl Hmilton syringe (30 S needle) ttched to syringe pump (KD Scientific, New Hope, PA) into the right CA1 of the hippocmpus (nteroposterior (AP): 3.8 mm; mediolterl (ML): 2.4 mm; dorsoventrl (DV): 3.0 mm, reltive to the regm), ccording to the tls of Pxinos nd Wtson. 43 After the injection, the needle ws left in plce for n dditionl 5 minutes efore eing slowly retrcted. A virl vector suspension in volume of 2.0 μl ws injected t 0.1 μl/minute over 20 minutes. Intrhippocmpl BDNF neutrlizing ntiody injection nd thromin injection. BDNF neutrlizing ntiodies (100, 200, or 400 ng in 4 μl PBS s modified doses of murine BDNF neutrlizing ntiodies 44 ; Snt Cruz, Snt Cruz, CA), thromin (20 U in 4 μl PBS; Sigm), nd mixture of BDNF neutrlizing ntiodies nd thromin were injected t rte of 0.5 μl/minute into the right CA1 of the hippocmpus (AP: 3.8 mm; ML: 2.4 mm; DV: 3.0 mm). Rts injected with neutrlizing ntiodies lone were used s control (Supplementry Figure S8). For western lot nlysis, rts received unilterl injection of 300 ng neutrlizing ntiodies ginst BDNF into the hippocmpl CA1 region t 3 weeks fter hrhe(s16h), nd the rins were hrvested 2 dys fter tretment (Supplementry Figure S9). All injections were mde using Hmilton syringe (30 S needle) ttched to syringe pump (KD Scientific). Rpmycin dministrtion. To investigte whether the production of BDNF fter hrhe(s16h) trnsduction of hippocmpl neurons is dependent on the ctivtion of mtorc1, nimls were posttreted with rpmycin (5 mg/kg, intrperitonel injection; LC Lortory, Wourn, MA), strting 3 weeks fter hrhe(s16h) injection nd continued dily until 4 weeks postinjection. The rin tissues treted with rpmycin or vehicle (4% ethnol, 5% Tween 80, nd 5% PEG400) 21 in the sence of hrhe(s16h) were used s controls. To clrify the effects of rpmycin on the sl level of BDNF expression nd mtorc1 ctivity in the hippocmpus, dditionl rts were treted with 10 mg/kg rpmycin for week nd then scrificed for western lot nlysis (Supplementry Figure S4). Immunohistochemicl stining procedures. Animls were trnscrdilly perfused nd fixed, nd rin sections (30 μm thick) were processed for immunohistochemicl stining s previously descried 29 with some modifictions. Briefly, rin sections (30 μm thick) were rinsed in PBS nd then incuted t 4 C with primry ntiody for 48 hours, nd then rin sections were rinsed with PBS 0.5% ovine serum lumin, incuted t room temperture with the pproprite iotinylted secondry ntiody, nd processed with n vidin iotin complex kit (Vector Lortories, Burlingme, CA). The signl ws detected y incuting sections in 0.5 mg/ml 3,3 -diminoenzidine (Sigm) in 0.1 mol/l PB contining 0.003% H 2 O 2. The stined smples were nlyzed under right-field microscope (Axio Imger, Crl Zeiss, Germny). The primry ntiodies were rit nti-flag (1:3,000; Sigm), rit nti-phospho-4e-bp1 (1:1,000; Cell Signling, Beverly, MA), mouse nti-neun (1:500; Millipore, Temecul, CA), nd rit nti-bdnf (1:200; Snt Cruz). For Nissl stining, rin sections were mounted on geltin-coted slides nd stined in 0.5% cresyl violet (Sigm), nd then nlyzed with right-field microscope (Axio Imger). For immunofluorescence leling, rin sections were rinsed nd incuted for 48 hour with one of the following pirs: rit nti-flag (1:3,000; Sigm) nd mouse nti-neun (1:500; Millipore) or mouse nti- NeuN (1:500; Millipore) nd rit nti-bdnf (1:200; Snt Cruz). Sections were then rinsed nd incuted with Cy3-conjugted ntimouse IgG or nti-rit IgG (1:200; Millipore), nd FITC-conjugted nti-rit IgG or ntimouse IgG (1:200; Millipore) for 1 hour, nd then wshed nd mounted with Vectshield mounting medium (Vector Ls, Burlingme, CA). The stined sections were viewed using confocl microscopy (LSM700, Crl Zeiss). Immunofluorescence stining for gli nd FLAG epitope. Brin tissues were prepred for immunohistochemicl stining s previously descried. 29 For immunofluorescence leling, while AAV--treted tissues (30 μm thick) were incuted with mouse nti-ox-42 (1:400; Serotec, Oxford, UK) or mouse nti-gfap (1:500; Sigm), AAV-hRhe(S16H)-treted sections were incuted with one of the following pirs for 48 hours: mouse nti-flag (1:2,000; Sigm) nd rit nti-i1 (1:2,000; Wko Pure Chemicl Industries, Osk, Jpn) or rit nti-flag (1:3,000; Sigm) nd mouse nti-gfap (1:500; Sigm). And then, sections were rinsed nd incuted with Cy3-conjugted ntimouse IgG (1:200; Millipore) nd FITC-conjugted ntirit IgG (1:200; Millipore), or with Cy3- conjugted ntirit IgG (1:200; Millipore) nd FITC-conjugted ntimouse IgG (1:200; Millipore) for 1hour. The stined sections were viewed using confocl microscopy (LSM700; Crl Zeiss). Western lot nlysis. Hippocmpl tissues were prepred from the nimls t 4 weeks fter injection of virl vectors s previously descried with some modifictions. 29 The tissues were homogenized nd centrifuged t 4 C for 15 minutes t 14,000g. The superntnt ws trnsferred to fresh tue, nd the concentrtion ws determined using BCA kit (Bio-Rd Lortories, Hercules, CA). The smples were oiled t 100 C for 5 minutes efore gel loding, nd equl mounts of protein (50 μg) were loded into ech lne with the loding uffer. Proteins nlyzed y gel electrophoresis were trnsferred to polyvinylidene difluoride memrnes (Millipore, Bedford, MA) using n electrophoretic trnsfer system (Bio-Rd Lortories), nd then, the memrnes were incuted overnight t 4 C with specific primry ntiodies. The following primry ntiodies were used: mouse nti-β-ctin (1:5,000; Acm, Cmridge, UK), rit nti-phospho-4e-bp1 (1:1,000; Cell Signling), rit nti-4e-bp1 (1:1,000; cell Signling), rit ntiphospho-p70s6k (1:1,000; Cell Signling), rit nti-p70s6k (1:1,000; Cell Signling), rit nti- (1:2,000; Millipore), mouse nti-flag (1:4,000; Sigm), nd rit nti-bdnf (1:500; Snt Cruz). After wshing, the memrnes were incuted with secondry ntiodies (Amershm Biosciences, Pisctwy, NJ) for 1 hour t room temperture, nd the lots were finlly developed with the ECL western-lotting detection regents (Amershm Biosciences). For semi-quntittive nlyses, the density of the immunolot nds ws mesured with Computer Imging Device nd ccompnying softwre (Fuji Film, Tokyo, Jpn). Mesurement of totl choline nd cetylcholine in the rt hippocmpus. The quntittive levels for totl choline nd cetylcholine were mesured y commercil colorimetric/fluorimetric kit ccording to the mnufcturer s instructions (Acm, Cmridge, UK; Supplementry Figure S2 nd S6). Briefly, the hippocmpl tissues were prepred from the nimls t 4 weeks fter intrhippocmpl injection of AAV- or AAV-hRhe(S16H), nd the tissues were homogenized nd centrifuged. The superntnt ws trnsferred to fresh tue, nd 50 μl of the ech smple ws mixed with 50 μl of rection solution including choline ssy uffer, choline proe, enzyme mix, nd cetylcholinesterse. The stndrd curve, ccording to the colorimetric procedure s indicted y mnufcturer s instructions, ws otined y diluting the choline stndrd. The mesurement ws otined y SoftMx softwre (Moleculr Devices, Sunnyvle, CA) t 570 nm. Moleculr Therpy vol. 23 no. 3 mr

10 Counting of hippocmpl CA1 neurons. As previously descried, 29 the numer of CA1 neurons ws counted in the rt hippocmpus. Briefly, lternte sections were otined t 3.3, 3.6, 4.16, nd 4.3 mm posterior to the regm, nd two regions from ech level (eight regions for ech niml) were used to count cells in the CA1 region. The numer of neurons within the CA1 lyer ws counted using light microscope (Crl Zeiss) t mgnifiction of 400 nd expressed s the numer of CA1 neurons per millimeter of liner length s descried previously with some modifictions. 45 To mintin consistency cross nimls, rectngulr ox ( mm) ws centered over the CA1 cell lyer eginning 1.5 mm lterl to the midline, nd only neurons with norml visile nuclei were counted. For quntifiction of p-4e-bp1-positive neurons, cells with p-4e-bp1 immunorectivity (rown color) in Nissl-positive cells, which hve size of over 10 μm, were counted. The numer of neurons in the ipsilterl hippocmpus ws quntittively expressed s percentge compred to the contrlterl control. Cell re nlysis. The re of Nissl stined neurons in the CA1 of hippocmpus ws determined y use of the DP2-BSW progrm (Olympus, Tokyo, Jpn) on computer ttched to light microscope (BX51; Olympus), interfced with chrge-coupled device video cmer (DP20-5; Olympus). Alternte sections were otined t 3.3, 3.6, 4.16, nd 4.3 mm posterior to the regm, nd rectngulr ox ( mm) ws centered over the CA1 cell lyer in ech section, nd five neurons in ech section were rndomly selected to provide 20 neurons per rt rin. The men re of neurons in the ipsilterl CA1 ws expressed s percentge compred to the contrlterl control. Hippocmpl slice preprtion. Rts were nesthetized with ether nd decpitted. All niml experiments were performed ccording to the policies nd guidelines of the Institutionl University, Seoul, ROK, study protocol #KHUASP(SE) The rins were removed t 4 weeks fter the injection of AAV-hRhe(S16H) nd plced in ice-cold rtificil cererospinl fluid (ACSF) tht ws composed of the following (in mmol/l): 125 NCl, 3 KCl, 1.2 KH 2 PO 4, 1.2 MgSO 4, 25 NHCO 3, 10 dextrose, nd 2 CCl 2 nd uled with 95% O 2 5% CO 2. Trnsverse hippocmpl slices (350 μm) were prepred with virting microtome (VT-1000S; Leic Biosystems Nussloch, Nussloch, Germny) in ice-cold ACSF. Slices were then mintined in recording chmer t room temperture for t lest 1 hour. All solutions were continuously uled with 95% O 2 nd 5% CO 2. LTP recording. Hippocmpl slices were trnsferred to recording chmer nd continuously perfused with ACSF (21 23 C). The Schffer collterl/commissurl-ca1 pyrmidl neuron responses were induced y stimultion of the Schffer collterl/commissurl pthwy with concentric ipolr electrode (200 μm dimeter; FHC, Bowdoin, ME). Extrcellulr recordings were otined with glss micropipette tht ws filled with 3 mol/l NCl (2 3 MΩ). The recording electrode ws plced long the trjectory of the Schffer collterl/commissurl pthwy. The stimulus intensity ws set to produce ~30% ( μv) of the mximum field excittory postsynptic potentil (fepsp). CA1 LTP ws induced y tetnic stimuli t 100 Hz for 1 second. Field EPSP mplitudes were verged over 10-second intervls nd expressed s percentges of the men fepsp mplitude tht ws mesured during the 30-minute seline period perfused with ACSF, which ws expressed s 100%. Behviorl test for seizures. To exmine whether the cellulr morphologic chnges induced y hrhe(s16h) in the dentte grnulr neurons cused ehviorl chnges, such s seizures, the nimls were recorded y video monitoring s previously descried 46 with some modifictions. Rts were nesthetized with chlorl hydrte (360 mg/kg; Sigm), nd they received unilterl intrhippocmpl injection of kinic cid (0.2 μg in 4 μl PBS; Sigm) using 10 μl Hmilton syringe (30 S needle) ttched to syringe pump (KD Scientific) into the right CA1 of the hippocmpus (AP: 3.8 mm; ML: 2.4 mm; DV: 3.0 mm, reltive to the regm). After the injection, the needle ws left in plce for n dditionl 5 minutes efore eing slowly retrcted. Recordings of spontneous seizures were strted 3 weeks fter hrhe(s16h) or kinic cid tretment, nd continued for 4 dys. The rts were monitored dily for 8 hours in order to evlute the ehviorl seizures. The seizure stge ws determined s previously descried 47 : stge 1, fcil movement; stge 2, wet dog shke; stge 3, forelim clonus; stge 4, rering; stge 5, rering nd flling; nd stge 6, jumping. The numer of seizures ws counted in the rts showing recurrent seizures. Sttisticl nlysis. Differences etween the two groups were nlyzed y the Student s t-test. Multiple comprisons etween groups were performed y one-wy nlysis of vrince nd Student Newmn Keuls nlysis. All sttisticl nlyses were performed using Sigm Stt Softwre (Systt Softwre, Sn Lendro, CA). SUPPLEMENTARY MATERIAL Figure S1. Nontrnsduction of hippocmpl microgli nd strocytes y AAV- or AAV-hRhe(S16H) in norml dult SD rts. Figure S2. The levels of totl choline (tch) nd cetylcholine (ACh) in the rt hippocmpus. Figure S3. Cytorchitecturl chnges in the grnule cell lyers y hrhe(s16h) trnsduction nd ehviorl tests for seizures. Figure S4. Effects of rpmycin on the hrhe(s16h)-incresed mtorc1 ctivity. Figure S5. Thromin cuses neuronl cell deth in the hippocmpus. Figure S6. hrhe(s16h)-incresed tch nd ACh re preserved in the thromin-treted hippocmpus. Figure S7. Thromin reduces mtorc1 ctivity in the hippocmpl neurons. Figure S8. Tretment with BDNF neutrlizing ntiodies (BDNF NA) hs no neurotoxicity. Figure S9. BDNF neutrliztion ttenutes the hrhe(s16h)- incresed mtorc1 ctivity in the hippocmpus. ACKNOWLEDGMENTS This reserch ws supported y the Ntionl Reserch Foundtion of Kore grnt funded y the Koren government (no. 2012R1A1A nd ). The uthors declred no conflict of interest. M.-T.J., J.H.N., W.-H.S., B.K.J., nd S.R.K. plnned nd crried out the experiments, nlyzed the dt, nd generted the figures. S.R.K., N.K., nd R.E.B. designed the virl vectors. M.-T.J., J.H.N., E.L., nd U.J.J. crried out immunohistochemicl stining. M.-T.J., J.H.N., nd S.-G.L. crried out western lot nlysis. S.R.K., K.H.J., nd Y.-S.B. nlyzed ehviorl dt. Y.-H.J. mesured the chnge of long-term potentition. 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