Effects of Estrogen on Beta-Amyloid-Induced Cholinergic Cell Death in the Nucleus Basalis Magnocellularis

Transcription

1 Pgintion Instrutions: This rtile should begin on RIGHT pge! Originl Pper Neuroendorinology DOI: / Reeived: August 18, 21 Aepted fter revision: September 8, 21 Published online: Otober 9, 21 Effets of Estrogen on Bet-Amyloid-Indued Cholinergi Cell Deth in the Nuleus Bslis Mgnoellulris Év M. Szegő Attil Csorb b Tmás Jnáky b Ktlin A. Kékesi István M. Ábrhám Gábor M. Mórotz Botond Penke b Miklós Plkovits d Ünige Murvi e Miklós S.Z. Kellermyer e József Krdos f Gábor D. Juhász Lbortory of Proteomis, Eötvös Loránd University, Budpest, b Medil Chemistry Deprtment, University of Szeged, Szeged, Hungry; Deprtment of Neurodegenertion nd Restortive Reserh, Georg-August University, DFG Reserh Center Moleulr Physiology of the Brin (CMPB), Göttingen, Germny; d Neuromorphologil nd Neuroendorine Reserh Lbortory, Deprtment of Antomy, Histology nd Endorinology, Semmelweis University nd the Hungrin Ademy of Sienes, e Deprtment of Biophysis nd Rdition Biology, Semmelweis University, nd f Deprtment of Biohemistry, Eötvös Loránd University, Budpest, Hungry Key Words Alzheimer disese Estrogen Amyloid Miropunh Proteomis MAPK pthwy Differentil two-dimensionl gel eletrophoresis Nuleus bslis mgnoellulris Abstrt Alzheimer disese is hrterized by umultion of -myloid (A ) nd ognitive dysfuntions linked to erly loss of holinergi neurons. As estrogen-bsed hormone replement therpy hs benefiil effets on ognition of demented ptients, nd it my prevent memory impirments, we investigted the effet of estrogen-pretretment on A -indued holinergi neurodegenertion in the nuleus bslis mgnoellulris (NBM). We tested whih A speies indues the more pronouned holinotoxi effet in vivo. We injeted different A ssemblies in the NBM of mie, nd mesured holinergi ell nd ortil fiber loss. Spheril A oligomers hd the most toxi effet. Pretretment of ovrietomized mie with estrogen before A injetion deresed holinergi neuron loss nd prtly prevented fiber degenertion. By using proteomis, we serhed for proteins involved in estrogen-medited protetion nd in A toxiity 24 h following injetion. The hnge in expression of, e.g., DJ-1, NADH ubiquinone oxidoredutse, ATP synthse, phosphtidylethnolmine-binding protein 1, protein phosphtse 2A nd dimethylrginine dimethylminohydrolse 1 support our hypothesis tht A indues mitohondril dysfuntion, dereses MAPK signling, nd inreses NOS tivtion in NBM. On the other hnd, ltered expression of, e.g., MAP kinse kinse 1 nd 2, protein phosphtse 1 nd 2A by A might inrese MAPK suppression nd NOS signling in the ortil trget re. Estrogen pretretment reversed most of the hnges in the proteome in both res. Our experiments suggest tht regultion of the MAPK pthwy, mitohondril ph nd NO prodution my ll ontribute to A toxiity, nd their regultion n be prevented prtly by estrogen pretretment. Introdution Copyright 21 S. Krger AG, Bsel Alzheimer disese (AD) is the most prevlent ge-relted neurodegenertive disese, nd the most ommon form of dementi [1]. Besides the umultion of -m- Fx E-Mil krger@krger.h 21 S. Krger AG, Bsel /1/ $26./ Aessible online t: Ev M. Szego Deprtment of Neurodegenertion nd Restortive Reserh Georg-August University, DFG Reserh Center Moleulr Physiology of the Brin Wldweg 33, DE 3773 Göttingen (Germny) Tel , Fx , E-Mil uni-goettingen.de

2 yloid (A ) peptide, neurofibrillry tngles nd synpti loss [2], nother hrteristi of AD is the erly degenertion of the bsl forebrin holinergi system (BFC) [3, 4]. Neurons of BFC express the reeptor 7 niotini etylholine reeptor, whih binds A seletively nd with high ffinity, mking holinergi neurons vulnerble in AD or proteting ginst A [5 7]. AD is known to be gender dependent nd ge-relted loss of estrogen (17 -estrdiol, E2) probbly inreses the risk of AD [8]. E2-bsed hormone replement therpy (ERT) deresed the risk of AD nd ognitive dysfuntions in some linil studies, suggesting protetive role of E2 [9 12]. Supporting these findings, E2 ws found to be neuroprotetive in in vitro nd in vivo experimentl AD models [13 16]. It is known tht E2 destbilizes A fibrils in vitro [17] nd dereses A lod in trnsgeni mie [18]. In ddition, E2 redues A -indued C 2+ overlod [19] nd p38 mitogen-tivted protein kinse (p38mapk) tivtion [2] in vitro. E2 is ble to optimize brin metbolism, hieving optiml energy onsumption during different mentl funtions [21 23]. Moreover, E2 tretment improves ognitive performne prtly vi intertion with the bsl forebrin holinergi system [24, 25]. However, despite the benefiil effets of E2 found in niml studies or reported from humn trils, some studies demonstrted tht ERT hs no effet on ognitive performne nd on severity of dementi in femle ptients [26, 27], mening tht ERT is still ontroversil for the tretment of dementi. B ot h A nd E2 generte widespred hnges in the brin by ontrolling gene expression mong others [22, 28 31], induing ell deth or prepring ells for toxiity nd ltering ellulr retions to A. In our present study, by serhing for possible trget proteins, we imed to find the mehnisms nd rise hypothesis underlying A toxiity on holinergi neurons nd the possible neuroprotetive effets of E2 in vivo. Hene, we first determined the neurotoxi potentil of differently ggregted A 1 42 solutions on the mouse nuleus bslis mgnoellulris-substnti innomint (NBM-SI) holinergi neurons in vivo. We mesured holinergi ell loss in the NBM-SI, nd holinergi fiber density in the projetion re (somtosensory ortex, SSCTX, lyer V). After seletion of the most potent A solution, in the seond prt of our experiments, we investigted the effet of E2 pretretment on A holinotoxi effets. In ddition, we performed proteomi investigtion to find the proteins influened by A nd A fter E2 pretretment. In this study, we present the possible mehnisms explining A toxiity nd the protetive effets of E2 ginst A on NBM-SI holinergi neurons. Methods Animls Animl breeding nd experiments were performed bsed on the rules of the Lol Animl Cre Committee t the Eötvös Loránd University, in ordne with the Europen Union onforming to the Hungrin At of Animl Cre nd Experimenttion. Femle wild-type C57BL6/J mie were mintined under 12-hour light/drk yle t 2 C, nd were supplied with wter nd food d libitum. In vitro Chrteriztion of A Solutions: Atomi Fore Mirosopy nd Thioflvin T Mesurements A 1 42 ( gift from Márt Zrándi [32] ) peptide ws dissolved in 1% hexfluoro-isopropnol (HFIP) for 6 h to prepre monomer solution [33]. The monomer solution ws entrifuged t 15, g (1 min), nd the superntnt ws lyophilized nd kept t 8 C until use. The morphology nd size of the A ggregtes in vitro ws determined using tomi fore mirosopy (AFM). A smples t 6 M onentrtion in gluose-free rtifiil erebrospinl fluid (ACSF, in m M : 144 NCl; 3 KCl; 1 MgCl 2 ; 2 CCl 2 ; ph 7.3) were inubted for, 12, 24 nd 48 h t room temperture. As the smples with 24 nd 48 h inubtion time did not dhere suffiiently to the mi surfe (Eletron Mirosopy Sienes, Htfield, P., USA), they were diluted in deionized wter (15- to 3-fold) nd the solutions were then inubted on the mi surfe for 2 min nd repled with wter. These solutions were mesured in wter insted of ACSF. Imges were obtined under similr onditions with n MFP3D AFM instrument (Asylum Reserh, Snt Brbr, Clif., USA), operting in nonontt mode, using the Olympus BioLever silion nitride ntilever (Olympus Co., Jpn); typil resonne frequeny: 9 khz. Drive mplitude nd ontt fore were kept to minimum. Ares were snned t.5 1 Hz rte. Imges were evluted by mesuring the height of 1 2 individul ggregtes bove the mi surfe using MFP-3D softwre (Asylum Reserh). Objets lerly ssoited from lrge number of ggregtes were tken s smple preprtion rtefts nd were exluded from the mesurements. It is importnt to note tht AFM mesurements my underestimte the dimeter of A strutures beuse of smple ompression by the AFM probe [34]. However, the tehnique is exellent to follow the progress of ggregtion nd the hnge in morphology. For thioflvin T (ThT) fluoresene mesurements, 1- l liquots were tken from the smples nd mixed with 1. ml of 5 M ThT (Sigm) in 5 m M glyine-noh buffer t ph 8.5 [35]. ThT fluoresene ws monitored immeditely t 485 nm with exittion t 445 nm in ell thermosttted to 25 C using SPEX FluoroMx instrument (Jobin-Yvon, Longjumeu Cedex, Frne). Exittion nd emission bndwidths were set to 5 nm. Dt re from 5 mesurements/time point. Preprtion of Tissue Setions for Toxiity Mesurements of A Assemblies To prepre different A ggregtes suitble for in vivo experiments, the lyophilized smples were dissolved in ACSF t onentrtion of 6 M nd kept t room temperture for, 12, 24 or 48 h prior to dministrtion. The onentrtion of the A solution ws determined in preliminry experiment, nd we seleted the lowest onentrtion to hieve minimum of 25% holin- 2 Neuroendorinology Szegő et l.

3 ergi ell deth (dt not shown). In order to eliminte the possible interferene of endogenous estrogen with A toxiity, dult femle 45- to 6-dy-old mie were bilterlly ovrietomized (OVX) under deep nesthesi using Avertin (2% 2,2,2-tribromoethnol, 1,2% myl-hydrte; 8% ethnol in physiologil sline; Sigm, USA). In the first set of experiments, we imed to determine the holinotoxi effet of different A ssemblies. A ws inubted in ACSF for, 12, 24 or 48 h t room temperture prior to injeting. On post-ovx dy 14, 2! 1 l 6 M A solution or ACSF ws injeted slowly into the NBM-SI using fused sili pillry (oordintes: L 2/+2, AP,65, DV 4,1/ 4,3) [36]. During stereotti surgery, mie were in deep nesthesi (1.5% hlothne in ir, 1.8 liters/min flow rte). ACSF ws injeted rndomly into one side, nd A solution into the other side of the brin; therefore, the sme niml served s its own ontrol (ACSF-injeted site, n = 5/group). On post-ovx dy 29, mie were nesthetized by lethl dose of Avertin, nd trnsrdilly perfused with 4% prformldehyde (Merk, Germny), ph 7.6 in phosphte-buffered sline solution. Brins were removed, postfixed for 2 h t 4 C nd ryoproteted in Tris-phosphte-buffered solution (TBS), ph 7.6, ontining 3% surose overnight t 4 C. 3- m oronl setions were ut on freezing mirotome nd four sets of setions were olleted in TBS. Determintion of the Effet of E2 on A Toxiity in vivo To study the effets of E2 pretretment on the A -exerted holinotoxiity, the following proedures were used. 45- to 6-dyold mie were bilterlly ovrietomized (see bove setion). On post-ovx dy 14, nimls reeived subutneous injetion of 1 g of 17 -estrdiol (E2; in.1 ml ethyl-olete vehile, Sigm) or the sme volume of vehile (EO). Twenty-four hours fter the EO/ E2 tretment, the previously prepred A solution (2! 1 l, 6 M in ACSF, 24-hour inubtion t room temperture) or ACSF ws injeted slowly into the NBM-SI (see the previous setion). Altogether, 12 mie were treted with EO nd A nd 12 mie with E2 nd A. Six EO-A -injeted nd 6 E2-A -injeted mie were used for differentil two-dimensionl gel eletrophoresis (DIGE) experiments (n = 6). These mie were srified 24 h fter the A -injetion (post-ovx dy 16) by ervil dislotion, brins were rpidly removed (! 4 s) nd frozen in dry ie. Seril 1- m oronl ryosetions were ut t 1 C nd tissue smples orresponding to the SSCTX nd the NBM-SI were disseted using the punh tehnique [37] ording to mirodissetion mp [38]. Smples were stored t 8 C until use. Six EO-A -, nd six E2-A -injeted mie were used for the regenertion study (n = 6). These mie were srified on post-ovx dy 3, perfused, nd their brins treted s desribed bove. Protein Isoltion, Differentil Two-Dimensionl Gel Eletrophoresis nd In-Gel Digestion Eight different sets of tissue smples were olleted for proteomi studies: four from EO-treted mie (SSCTX nd NBM-SI from the ACSF-injeted side nd SSCTX nd NBM-SI from the A -injeted site), piees of tissue from 6 different nimls, therefore 6 piees/re/tretment group, nd the sme res from the E2-treted nimls (6 mie). Tissue smples were homogenized s we reported erlier [23]. The ph of the superntnt orresponding to ytosoli nd membrne frtions ws djusted to 8.. Five mirogrm of eh protein smple ws lbeled with Cy5 sturtion dye CyDye DIGE Fluor-Lbeling Kit for Sre Smples (4 nmol/ 5 g protein, GE Helthre, Uppsl, Sweden) ording to the mnufturer s instrutions. The referene smple (internl stndrd, equl mounts: 5 g of protein from ll E2- nd ll EOtreted smples from the sme region) ws lbeled with Cy3 nd the two differently mrked smples (5 g of Cy5-lbeled smple nd 5 g Cy3-lbeled referene) were multiplexed to be resolved in the sme gel. Lbelled proteins were seprted nd visulized s desribed previously (DryStrip ph: 4 7, 1% rylmide gel) [23]. Differentil protein nlysis ws performed using DeCyder softwre pkge 6., DIA nd BVA modules (GE Helthre). For the identifition of proteins in spots of interest, preprtive 2D eletrophoresis ws performed seprtely using 8 g of proteins per gel [23]. LC-MS Anlysis nd Protein Identifition nd Extended Literture Serh LC-MS nlysis ws performed by n Agilent HPLC-Chip/MS system onsisting of 11 Series HPLC system nd 633 LC/ MSD XCT Plus ion trp mss spetrometer. The LC system ws operted in smple enrihment/deslting mode using ProtID- Chip-43 (II Chip-Column) (olumn mteril: ZORBAX 3 SB- C18 5 m, trp olumn volume: 4 nl, nlytil olumn: 43! ID.75 mm). The HPLC solvents were the following: solvent A:.1% formi id in wter nd solvent B:.1% formi id in etonitrile. Two miroliter of smple ws enrihed with solvent A t flow rte of 4 l/min for 2 min using pillry pump then followed by grdient elution of trypti peptides t flow rte of 3 nl/min using nno pump. The grdient ws 5% B/min inrese of onentrtion B from 5 to 45% under 1 min followed by grdient olumn-wsh from 6 to 9% solvent B in 2 min. The mss spetrometer ws operted in the utomsms mode, the resolution ws less thn.5 u (FWHM) nd the sn speed ws 8,1 u/s. The survey sn rnge ws 3 1,6 m/z nd 4 ions were seleted for CID. MSMS sn rnge ws set to 1 18 m/z t 26, u/s t resolution of less thn.6 u (FWHM). All quired MSMS dt were proessed nd subjeted to dtbse serh by Agilent Spetrum Mill MS Proteomis Workbenh Ver. A.3.2 ginst NCBInr (11/16/29 dtbse). Dtbse serh prmeters were set to 1.5 D for preursor ion mss tolerne,.6 D for frgment ion mss tolerne. Constnt modifition of Cys ws speified s rbmidomethyltion, vrible modifitions were set to methionine oxidtion, deminted glutmine, etyltion of protein N-terminl nd oxidtion of tryptophn. For protein ssignment the minimum sore ws set to 2 for proteins nd peptide hits were epted bove sore of 9, 1, 11 nd 12 ording to their hrge stte of 1+, 2+, 3+ nd 4+, respetively [39]. To mke funtionl interprettion of our dt, systemti literture survey ws onduted [23]. Immunohistohemistry nd Aetylholinesterse Histohemistry To detet holinergi neurons, free-floting, peroxidse-bsed immunohistohemistry ws performed in the sme mnner we reported previously with only slight modifition [4]. We used ntiholine etyl-trnsferse (nti-chat ntibody (1: 2), Snt Cruz Biotehnology, Clif., USA) for 48 h t 4 C. For fluoresent lbeling of lbindin (1: 3,, Swnt, Switzerlnd) nd ChAT (1: 1,), we used floresent dye-onjugted seondry ntibodies (1: 1,, AlexFluor-488 for ChAT nd AlexFluor-555 for lbindin; Invitrogen, Clif., USA). Visuliztion of etyl- E2 Redues A -Indued Neuron Loss in the NBM Neuroendorinology 3

4 holinesterse fibers ws done ording to the method of Hedreen et l. [41]. Dt Anlysis nd Sttistis Number of ChAT-ir ells were ounted by n Olympus BX51 mirosope (Olympus Optil, Hmburg, Germny) using! 4 objetives. Every fourth setion ws stined nd ChAT-ir neurons of the NBM-SI were ounted from both sides (ACSF- or A -injeted sides from four setions) by n investigtor blind to the experimentl groupings; finlly, the number of neurons/ m 3 nd the perentge hnge ws determined. Dt re expressed s men 8 SEM. ANOVA ws rried out to exmine the effets of EO/E2 nd ACSF/A tretment (R softwre pkges, version 2.8., R Development Core Tem 28, Vienn, Austri). Aetylholinesterse (AChE) fiber density ws determined in the SSCTX, lyer V. As the holinergi neurons of the NBM projet lmost exlusively unilterlly to the SSCTX, it llowed us to determine the differenes between ACSF- nd A -injeted sides in the sme niml. Every fourth setion ws stined nd six setions were nlyzed per niml. Six imges were tken of every slie using! 1 objetive (Olympus BX51) nd the re overed by AChE fibers ws mesured ten times from the sme imge following bkground subtrtion nd binriztion. Therefore, six setions/niml, six imges/setion nd 1 mesurements/imge resulted in hierrhil nested design. The effet of A on fiber density ws expressed s perentge hnge reltive to the ACSF-injeted side. A generlized liner mixed model ws pplied, the dependent vrible ws the fiber density, nd the independent vribles were the EO or E2 tretment nd ACSF or A injetion. The identifition of nimls, setions nd imges were set s rndom ftors (R softwre). For the DIGE experiments, ANOVAs were lulted by the DeCyder softwre Biologil Vrine Anlysis (BVA module). The internl stndrd ws pool of equl mounts of ll smples (from ll SSCTX or ll NBM-SI within the experiment); it ws representtive of every protein present nd ws the sme ross ll gels from the sme region. The stndrd provided n verge imge ginst whih ll other gel imges were normlized, removing muh of the experimentl vrition nd reduing gel-to-gel vrition. R e s u l t s In vitro Chrteriztion of A (1 42) Aggregte Size nd Morphology Smples with h inubtion time minly ontined prtiles exhibiting height not exeeding 1 nm (pproximtely 73%; fig. 1, b). This size rnge possibly orresponds to the monomer stte of A, while prtiles lrger thn 1 nm ould be ssigned to the ggregted forms of A peptide [33]. The perentge of the smll prtiles signifintly deresed with inubtion time to pproximtely 5, 25 nd 5% fter 12, 24 nd 48 h, respetively ( fig. 1, b). We observed greter portion of lrge prtiles fter longer inubtion times. After 12 h inubtion, the rtio of prtiles with 1 2 nm height inresed, form orresponding to smll oligomers [33]. Aggregtes formed in the 2 4 nm rnge ppered fter longer inubtion times (24 nd 48 h) with mximum ourrene t nm ( fig. 1, b). We observed hnge in the morphology of the ggregtes prlleling their inresed size. Spheril ggregtes ppered in the 24-hour smples ( fig. 1 b), while fter 48 h, besides spheril oligomers, short, fibrillr strutures were formed with 5 2 nm length nd verge height of nm ( fig. 1 b). The ltter speies of ggregtes ould not hve been mture myloid fibrils beuse of their shortness nd smll dimeter. Rther, these were protofibrils formed from oligomers. Thioflvin T is fluoresent dye tht binds to protein ggregtes, espeilly to myloid fibrils, while it does not bind to monomers. Upon binding, it shows high fluoresene intensity. -hour smples exhibited low ThT fluoresene intensity refleting the high monomer ontent in the solution ( fig. 1 ). 12-, 24- nd 48-hour smples exhibited n inresing ThT fluoresene intensity with time inditing the progress of the ggregtion proess nd n inrese in the myloid-like struture ontent (fig. 1). Determintion of the Cholinotoxi Potentil of Different A Forms Injetion of the -hour solution hd no effet on the number of holinergi ells in the NBM-SI ( fig. 2 ) nd filed to indue holinergi fiber loss in the SSCTX ( fig. 2 b). In ontrst, the holinotoxi potentil of the different A solutions inresed with pre-inubtion time (loss of neurons = ounted ontrol site-ounted lesioned site; 12-hour solution: pproximtely 15% ell loss, p =.33; 24-hour solution: pproximtely 25% ell loss, p =.2; 48-hour solution: pproximtely 26% ell loss, p =.2) lthough the 48-hour smple hd the sme effet s the 24-hour smple (p =.679; fig. 2 ). Similrly, fiber Fig. 1. In vitro hrteriztion of size distribution nd morphology of A 1 42 ggregtes. Representtive AFM imges of A smples fter inubtion for, 12, 24 nd 48 h, respetively. Sle brs represent 2 nm. Color odes for height tres re presented. b Height distributions of A smples (AFM mesurements) fter different inubtion times t room temperture. Columns represent the perentge of individul ggregtes of ertin height.2 nm intervl. Lrge objets 1 6 nm were exluded from lultion. Chnges in the intensity of thioflvin T fluoresene show inresing ggregtion of A 1 42 with time. ThT binds protein ggregtes, but not monomers. Dt re from 5 mesurements/time point. 4 Neuroendorinology Szegő et l.

5 1 b 3 h h h 2 1 Abundne (%) h 12 h h Height (nm) h 2 1 ThT fluoresene (AU) Inubtion time (h) h E2 Redues A -Indued Neuron Loss in the NBM Neuroendorinology 5

6 Number of ChAT-ir neurons 2,5 2, 1,5 5 b Reltive AChE fiber loss (%) b ACSF h 12 h 24 h 48 h ACSF h 12 h 24 h 48 h A solutions inubted for different times b A solutions inubted for different times 2,5 3 Number of ChAT-ir neurons 2, 1,5 5 b Reltive AChE fiber loss (%) b EO E2 EO E2 ACSF EO E2 ACSF A d 24-hour A solution Fig. 2. Effets of inubtion time of A solution nd E2 pretretment on unilterl NBM lesions indued by A., b The toxi effet of the 6 M A solution inreses with preinubtion time. Quntittive nlysis of holinergi ell number ChAT-ir ( ) nd re frtion overed by holinergi AChE-positive fibers ( b ) reveled n inresing toxi effet of A solution with inresing inubtion time. AChE fiber density ws mesured in the lyer V of the SSCTX, nd fiber loss is presented s reltive perentge of the ACSF-injeted ontrlterl side. Mximum neuron nd fiber loss ws found fter 24 or 48 h inubtion time of A. 24 h E2 pretretment did not inrese holinergi ell numbers, s we found ompring the ontrlterl ACSF-injeted sides of EOtreted mie (open brs) nd E2-treted mie (digonl brs). E2 deresed the vulnerbility of holinergi ells to A lesion ompred to EO pretretment ipsilterl sides. d E2 pretretment signifintly deresed A -indued AChE fiber loss in the SSCTX. Different letters show signifint differenes, p!.5. Dt re expressed s men 8 SEM, n = 5 (, b ) or n = 6 (, d ). loss inresed in the SSCTX prllel with the inresing inubtion time of the A solutions (fiber loss = mesured ontrol SSCTX-mesured lesioned SSCTX; 12- hour solution: pproximtely 14% fiber loss, p =.14; 24-hour solution: pproximtely 23% fiber loss, p =.2; 48-hour solution: pproximtely 26% fiber loss, p =.2; fig. 2 b) nd the 24- nd 48-hour A solutions indued equl holinergi fiber loss (p =.153). Compring the size, morphology ( fig. 1 ) nd in vivo effet of the different A solutions the observed toxiity ( fig. 2, b) ould not be relted to monomers in our model. Moreover, the similr toxi effet of 24- nd 48-hour smples suggests tht the most effetive omponents re not mture ggregtes, sine thioflvin T binding (hene ggregtion) in- 6 Neuroendorinology Szegő et l.

7 resed with time ( fig. 1 ). Our dt lso indite tht toxi speies re not protofibrils sine these prtiles re missing from the 12- nd 24-hour smples ( fig. 1, b). Bsed on our in vivo nd in vitro experiments, we suggest tht the most toxi speies re not the protofibrils, but rther the spheril oligomer forms in the size rnge of 1 3 nm. Pretretment with 17 -Estrdiol Hs Protetive Effet ginst A We seleted the 24-hour inubtion time for the further experiments, s this A solution (nd the 48-hour solution) hd the more pronouned holinotoxi effet. Pre-tretment with EO vehile hd no effet on the A toxiity (ell loss fter A : pproximtely 23%, p =.1; fiber loss: pproximtely 25%, p =.9, ompred to the ACSF-injeted site; fig. 2, d). E2 signifintly deresed the ytotoxi effet of A ompred to EO tretment (pproximtely 15 vs. 23% ell loss, p =.26), lthough it did not eliminte the toxi effet (p =.3, omprison of E2- nd ACSF-treted sides). Prllel with deresed neuron loss, E2 pretretment redued A -indued holinergi fiber loss in the SSCTX (pproximtely 12 vs. 25% fiber loss, p =.6, fig. 2 d), but it ould not eliminte the A toxiity (p =.2, omprison of E2-ACSF nd E2-A ). The similr hnge in the A -indued ell nd fiber loss fter E2 pretretment suggests tht E2 hd no regenertive effet, rther neuroprotetive pity. Identifition of Differentilly Expressed Proteins fter A or E2-A Tretment In this experiment, we imed to find proteins nd possible erly pthwys influened by A or A +E2 tretment, right fter A injetion. Usully, hnges in the expression of struturl proteins our in suessive steps, but s delyed response. Therefore, we nlyzed protein expression pttern 24 h fter A injetion using differentil two-dimensionl gel eletrophoresis (DIGE) nd mss spetrometry. Altogether 1,44 spots were present on the mster gel from smples of the SSCTX, nd 1,723 from the NBM-SI s we determined using DeCyder softwre. Forty-eight hours fter E2 nd 24 h fter A tretment, the mjority of protein spots showed only smll hnges between groups. However, two-wy ANOVA ross ll the gels showed signifint differene in the intensity of 127 spots in the NBM-SI nd 95 in the SSCTX. We ould identify 56 proteins from the 127 spots of the NBM-SI smples nd 35 proteins from 95 spots of the SSCTX. Representtive 2-D gel mps nd 3D reonstrution of protein spots re shown in figure 3. Identified proteins, showing signifint differenes, re listed in tbles 1 nd 2. We used the EO vs. E2 nd ACSF vs. A smples s independent vribles in the nlysis nd ould thereby mke four resonble omprisons between groups: (1) EO-ACSF vs. EO-A, (2) E2-ACSF vs. E2-A, (3) ACSF vs. A, nd (4) EO-A vs. E2-A. Herefter, we onentrte on the omprison of nimls treted with (1) EO- A vs. EO-ACSF, s this omprison gives us informtion bout the mehnism of A toxiity (injeted into the NBM-SI) nd (4) EO-A vs. E2-A, s this lter nlysis my provide evidene bout the protetive effets of E2 ginst A. Regrding just these omprisons, we ould identify 42 protein hnges in the NBM-SI ( tble 1 ), nd 27 hnges in the SSCTX ( tble 2 ). We lustered the identified proteins into seven funtionl groups, nmely ntioxidnt defene, ytoskeleton, metbolism, protein turnover nd stbility, signling, synpti proesses nd unknown. We ould identify proteins from the NBM involved in the regultion of the redox homoeostsis ( tble 1 ): expression of protein disulfide isomerse ssoited 3 nd dimethylrginine dimethylminohydrolse 2 deresed, while level of DJ-1 protein inresed fter A injetion, suggesting deresed ntioxidnt level. In ontrst, 24- hour E2 pretretment reversed these hnges, nd inresed the expression of two further proteins (inner membrne protein nd glutthione S-trnsferse). A indued hnges in the expression of ytoskeletl proteins (dihydropyrimidinse-like 2, fsin nd -tin), while E2 prevented or reversed these hnges. Chnges in the proteins involved in generl ellulr metbolism were lso observed, nd A -indued hnges were prevented or reversed by E2 pretretment. Interestingly, A deresed expression of otubin-1 nd protesome 26S subunit, proteins involved in protein degrdtion, while E2 pretretment prevented these hnges. Proteins involved in intrellulr signling (e.g. protein phosphtse 2A) or C 2+ buffering (lbindin) were lso identified s trgets of A or E2. Proteins involved in ntioxidnt defense were identified from SSCTX smples (tble 2) fter A injetion. Expression of dimethylrginine dimethylminohydrolse 1 nd sepipterin redutse inresed, while protein disulfide isomerse ssoited 3 deresed fter A injetion. E2 pretretement reversed these hnges. Expression of some ytoskeletl proteins like dihydropyrimidinselike 2 nd septin 8 deresed, while -tin inresed fter A -injetion. E2 pretretment before A injetion E2 Redues A -Indued Neuron Loss in the NBM Neuroendorinology 7

8 b e d f d e f Fig. 3. Representtive 2D gel imges from the NBM-SI (,, d ) nd SSCTX ( b, e, f )., b Pseudo-olored 2D mps of NMB-SI nd SSCTX, respetively. The internl stndrd pooled from ll of the smples from the sme ntomil re ws lbeled with Cy3 green, smples from the tretment group A -EO were lbeled with Cy5 red. f 3D reonstrution of protein spots mrked in ( ) or ( b ) gel mps. Grphs represent the bundne of the s p ot re l t i ve to t he i nt e r n l s t nd rd. prevented these hnges, nd inresed the expression of septin 3 protein. A reltively low number of proteins ws identified from the metbolism groups, mening tht A hd lower effet on generl ellulr homeostsis outside of the injetion site. Interestingly, we found A -indued upregultion of polipoprotein A-1, while E2 pretretment deresed the expression of this protein. Proteins with hperon tivity (e.g. het shok protein 15 kd, stress protein 7) were lso ltered by A or E2!A tretment, moreover, expression of mropin subunit 28 inresed fter A injetion, but deresed with E2 A tretment. In ontrst to the findings from the NBM, we observed inresed MEKK1 nd deresed protein phosphtse 2A expression, suggesting the tivtion of ERK pthwys following A injetion. Seernin 1, protein involved in synpti proesses, deresed, while EH domin ontining 3 inresed in expression following A injetion, nd E2 tretment reversed these hnges. Deresed Clbindin Immunoretion in the NBM-SI fter A Injetion We seleted lbindin from the protein list to hek the expression pttern fter A injetion of EO- nd E2- treted mie. We found no ololiztion of lbindin 8 Neuroendorinology Szegő et l.

9 Tble 1. Proteins identified from NBM-SI fter A or E2-A tretment Spot No. Protein Aession No. EO-ACSF vs. EO-A EO-A vs. E2-A MW PI % Seq. p vlue Av. rtio p vlue Av. rtio Antioxidnt defense SI21 inner membrne protein, mitohondril SI482 protein disulfide isomerse ssoited SI1474 N(G),N(G)-dimethylrginine dimethylminohydrolse SI1633 DJ-1 protein SI1669 glutthione S-trnsferse, lph Cytoskeleton SI254 dihydropyrimidinse-like SI256 dihydropyrimidinse-like SI381 dihydropyrimidinse-like SI633 fsin SI886 gmm-tin Metbolism SI212 NADH-ubiquinone oxidoredutse 75-kD subunit, mitohondril SI433 retine kinse, brin SI625 ldhehyde dehydrogense fmily 5, subfmily A SI66 ldehyde dehydrogense fmily 7, member A SI665 enolse 2, gmm neuronl SI685 ATP synthse, H+ trnsporting, mitohondril F1 omplex, lph subunit, isoform 1 SI817 suinte-oenzyme A ligse, ADP-forming, bet subunit SI1191 ltte dehydrogense B SI1194 ltte dehydrogense B SI131 mlte dehydrogense 1, NAD (soluble) SI1414 tyrosine 3-monooxygense/tryptophn 5-monooxygense tivtion protein, polypeptide SI153 phosphoglyerte mutse SI1643 hloid dehlogense-like hydrolse domin ontining SI1677 ATP synthse, H+ trnsporting, mitohondril F1 omplex, lph subunit, isoform Protein turnover nd stbility SI169 het shok protein 1, lph SI72 protesome (prosome, mropin) 26S subunit, ATPse SI515 het shok protein 1 (hperonin) SI135 otubin Signling SI676 gunosine diphosphte (GDP) dissoition inhibitor SI1313 protein phosphtse 2A, regultory subunit B (PR 53), isoform CRA_b SI1467 inositol (myo)-1(or 4)-monophosphtse 1, isoform CRA_b SI1538 lbindin SI1548 lbindin SI1553 lbindin-28k, isoform CRA_ SI564 protein phosphtse 3, tlyti subunit, lph isoform, isoform CRA_d Synpti proesses SI122 Aldh1l1 protein SI5 phosphtidylethnolmine binding protein SI572 glutmte dehydrogense 1 preursor SI137 phosphtidylethnolmine binding protein SI1711 phosphtidylethnolmine binding protein Unknown SI1571 mcg961, isoform CRA_ SI1716 RIKEN DNA 11167D22, isoform CRA_ Nmes of proteins with ltered expression level fter A tretment re shown in blue, proteins hnged fter E2-A tretment re in blk nd proteins with ltered expression level fter both A nd E2-A tretments re in red. Av. rtio: rtios of protein expression levels were lulted using DeCyder softwre pkge, Biologil Vrine Anlysis module, s the fold hnge between normlized spot volume of EO-ACSF- nd EO-A -treted smples or between EO-A - nd E2-A -treted smples. Vlues below zero: deresed protein level fter the speifi tretment. Av. = Averge; MW = moleulr weight; PI = isoeletri point; MW nd PI s determined by Uniprot dtbse; % Seq.: protein sequene overge. E2 Redues A -Indued Neuron Loss in the NBM Neuroendorinology 9

10 Tble 2. Proteins identified from SSCTX fter A or E2-A tretment Spot No. Protein Aession No. EO-ACSF vs. EO-A EO-A vs. E2-A MW PI % Seq. p vlue Av. rtio p vlue Av. rtio Antioxidnt defense SCTX956 dimethylrginine dimethylminohydrolse SCTX161 sepipterin redutse SCTX387 protein disulfide isomerse ssoited Cytoskeleton SCTX137 dihydropyrimidinse-like SCTX181 dihydropyrimidinse-like SCTX196 dihydropyrimidinse-like SCTX413 septin 8, isoform CRA_ SCTX76 gmm-tin SCTX868 neuronl-speifi septin Metbolism SCTX96 mlte dehydrogense 1, NAD (soluble), isoform CRA_ SCTX871 isoitrte dehydrogense 3 (NAD+) lph, isoform CRA_ SCTX986 polipoprotein A-I Protein turnover nd stbility SCTX16 ubiquitin-tivting enzyme E1 isoform SCTX21 het shok protein 15kD SCTX117 stress-7 protein, mitohondril SCTX12 stress-7 protein, mitohondril SCTX369 het shok protein 1 (hperonin) SCTX461 stress-7 protein, mitohondril SCTX581 protesome (prosome, mropin) 28 subunit, lph Signling SCTX7 protein phosphtse 2A, regultory subunit B, delt isoform SCTX98 protein phosphtse 1, tlyti subunit, bet isoform SCTX38 protein phosphtse 1, regultory (inhibitor) subunit 1B SCTX63 gunosine diphosphte (GDP) dissoition inhibitor 2, isoform CRA_b SCTX692 mitogen tivted protein kinse kinse 2, isoform CRA_e SCTX734 mitogen tivted protein kinse kinse Synpti proesses SCTX54 dynmin SCTX271 N-ethylmleimide sensitive fusion protein tthment protein lph SCTX362 EH-domin ontining SCTX635 seernin SCTX148 synulein, lph, isoform CRA_b Nmes of proteins with ltered expression level fter A tretment re shown in blue, proteins hnged fter E2-A tretment re in blk, nd proteins with ltered expression level fter both A nd E2-A tretments re in red. Av. rtio: rtios of protein expression levels were lulted using DeCyder softwre pkge, Biologil Vrine Anlysis module, s the fold hnge between normlized spot volume of EO-ACSF- nd EO-A treted smples or between EO-A - nd E2-A -treted smples. Vlues below zero: deresed protein level fter the speifi tretment. Av. = Averge; MW = moleulr weight; PI = isoeletri point; MW nd PI s determined by Uniprot dtbse; % Seq.: protein sequene overge. nd ChAT signls in the ACSF sites (online suppl. fig. 1A, Injetion of A in EO-treted mie indued the loss of both ChAT nd lbindin-positive neurons (online suppl. fig. 1B). E2 pretretment ttenuted both ChAT nd lbindin-positive neuron loss from the NBM-SI (loss of lbindin-positive neurons in the NBM-SI of EO-treted mie fter A injetion: p =.17; in the E2-treted nimls: p =.461; but no signifint differene between EO- or E2- treted, A -injeted sides: p =.249). Interestingly, we 1 Neuroendorinology Szegő et l.

11 observed some lbindin-positive holinergi neurons in the E2-A -treted mie, but not in E2-ACSF- or EO-A treted nimls (online suppl. fig. 1C). Disussion The present study demonstrtes tht (1) A solutions of different omposition hve different toxi potentil on NBM holinergi neurons in vivo; (2) pretretment with E2 protets holinergi ells nd fibers ginst A toxiity, nd (3) A lone or in ombintion with E2 pretretment indues speifi hnges in the brin proteome of mie. S ph e r i l A 1 42 Oligomers Indue Cholinergi Cell Deth, but E2 Pretretment Is Protetive ginst A Toxiity There is no greement in the literture s to whih A form is the most toxi. Oligomers nd fibrils re well known ytotoxins in vivo [42 45], disrupting holinergi neurotrnsmission [46] ; however, the monomer ws found to be neuroprotetive in vitro [47]. In the present study, A solution ontining minly monomers ( h) hd no toxi effet on holinergi neurons ( fig. 1, 2 ). In ontrst, we observed inresing ytotoxiity with inresing rtios of prtiles with 1 2/2 3 nm height. Spheril A oligomers (observed t 24 nd 48 h) indued the mximl neuron loss lulted s the differene between the ontrol nd lesioned sites. On the other hnd, s the protofibrils were present in the 48-hour solutions but not in the 24-hour ones, we exlude them s the most toxi speies on holinergi neurons. It is importnt to note tht our dt do not exlude the possibility tht A might ffet other neurotrnsmitter systems. Indeed, injetion of A into the NBM indues hypofuntion of GA- BAergi neurons [48] nd disturbs the serotonergi innervtion of the rt bsl forebrin nd erebrl ortex [49, 5]. Moreover, s we found loss of lbindin signl in the NBM-SI, but not in holinergi neurons (online suppl. fig. 1), A indeed regulted lso other neurons, suh s glutmtergi or GABAergi ells [51, 52]. Although linil evidene is still ontroversil, it is well epted tht protetion by E2 my vry with dose nd timing of the tretment in vivo nd in vitro [53]. E2 provides protetion even two hours fter the insult by rpid tivtion of signling pthwys nd ntioxidnt mehnisms [54, 55]. Furthermore, E2 pretretment develops ellulr tolerne by induing gene trnsription nd protein synthesis [23, 56, 57]. This fine-tuning inludes hnges in metbolism nd synthesis of nti-poptoti nd ntioxidnt proteins [58]. In the present study, lthough single injetion of E2 ws not enough to inrese the bsl number of holinergi neurons or ortil fiber density [59], it ws ble to redue the toxi effet of A ( fig. 2, d). However, in ontrst to in vitro experiments by others [6], in our study E2 ould not eliminte ellulr A toxiity or indue further regenertion of ortil projetions ( fig. 2 d). Contrry to our dt, in nother study [59] 2-week E2 tretment of rts enhned ortil holinergi projetions but did not ffet the exitotoxiity-indued reltive lesion. However, in this study, prolonged E2 tretment more likely inreses regenertion thn single injetion 24 h before the A exposure. Moreover, injetion of N-methyl- D -sprtte (NMDA) in the NBM resulted in pproximtely 5% loss of holinergi neurons; while in our study, more moderte holinergi ell loss (pproximtely 25%) ws indued using A. In ddition, the most likely different toxi mehnisms indued by A nd NMDA n lso be responsible for the different results. Puttive Mehnisms of Neurotoxiity nd Neuroprotetion We hypothesized tht A nd E2 indue hnges in the proteomes tht medite ell deth or protet neurons. Comprison of proteomes of (1) EO-ACSF vs. EO-A, nd (2) EO-A vs. E2-A provides informtion bout the neurotoxi effet of (1) A nd (2) protetive mehnisms of E2 ginst A toxiity. We nlyzed protein hnges in the NBM-SI (ell bodies, injetion), nd hnges in the SSCTX (projetion). Herefter, the nmes of proteins with ltered expression re in bold print; see tbles 1 nd 2. Hypothetil pthwys suggested to be influened by A or E2-A tretment re depited in figure 4. A indued upregultion of NADH ubiquinone oxidoredutse, nd downregultion of ATP synthse ( tble 1 ; fig. 4 ) in the NBM-SI. Inresed NADH oxidtion nd deresed ATP prodution utilizing ph grdient might mke mitohondri idi, deplete ATP nd trigger poptoti signling [61]. Reently, Rhein et l. [62] hve found deresed mitohondril eletron trnsport omplex IV tivity, drop in ATP level nd upregultion of omplex I proteins in A -trnsgeni mie. These dt re in line with the deteted hnges in our experiment. On the other hnd, E2 pretretment prevented the A - indued regultion of these proteins, preserving mitohondril integrity. Mitohondril dysfuntion is often ssoited with inresed prodution of retive oxygen speies induing intivtion of proteins of the respir- E2 Redues A -Indued Neuron Loss in the NBM Neuroendorinology 11

12 b 12 Neuroendorinology Szegő et l.

13 Fig. 4. Shemti illustrtion of pthwys supposed to be regulted by A nd/or E2 tretment. We s s u me t h t A injetion in the NBM-SI inreses mitohondril idity NADH-dehydrogense, ATP synthse nd dereses DJ-1 expression. A might inhibit MAPK signling PP2A, PDEBP-1. E2 prevented the regultion of these proteins. b A injetion in the NMB indued deresed MAPK pthwy inhibition PP2A, MEK1 nd inresed NOS tivtion sepipterin redutse, dimethylrgininse-2. However, 24 h E2 pretretment rther led to deresed MAPK signling PP2A, PP1, RhoGDI MEK1, MEK2 nd inhibited NOS tivtion fter A tretment. Nmes of proteins with ltered expression re in bold, nd protein regultion by A is indited with blue rrow nd by E2 with red rrow. Continuous/dshed rrows indite diret/indiret regultion, respetively. UPS = Ubiquitin-protesome system; Hsp = het shok protein; PDIA = protein disulfide isomerse ssoited; ubi = ubiquitin; GST = glutthione S-trnsferse; NOS = nitrogen oxide synthse; ADMA = symmetri dimethylrginine; PEBP-1 = phosphtidylethnolmine binding protein 1; PP = protein phosphtse; RhoGDP DI = Rho-GDP dissoition inhibitor; MAPK = mitogen-tivted protein kinse; BH4 = tetrhydrobiopterin; MEK = M A P k i n s e k i n s e 1. tory hin, s it ws shown in vivo [62]. Moreover, oxidtive intivtion of neuronl proteins my led to the development of AD [63]. In our experiments, A deresed the level of DJ-1 protein, hperon whih protets mitohondril omplex I under oxidtive stress [64]. Moreover, A inresed the expression of dimethylrgininse-2, n enzyme tht hydrolyses symmetri dimethylrginine (ADMA) [65]. As ADMA is n endogenous inhibitor of nitrogen monoxide synthse [66], A ould trigger NO prodution nd indue further oxidtive dmge. E2, in turn, inresed DJ-1, deresed dimethylrgininse-2 nd inresed glutthione S-trnsferse expression ( fig. 3 ). Neurons re prtiulrly suseptible to NO nd peroxynitrite exposure, nd the nitrostive stress my depend on the level of redued glutthione [67]. Therefore, E2 pretretment might redue nitrostive stress in the NBM. Signling pthwys ssoited with rf kinses plys role in neuronl survivl nd deth signling. We found upregultion of two rf kinse inhibiting proteins following A tretment, nmely phosphtidylethnolminebinding protein 1 or Rf kinse inhibitor protein (RKIP) nd protein phosphtse 2A (PP2A) [68, 69]. The possible intivtion of ERK1/2, p38 or JNK pthwys is in ontrst to some studies reporting A -indued pthologil tivtion of rf pthwys [7, 71]. Contrry to our work, A indued rpid ERK1/2 phosphoryltion in vitro [71], or delyed (48 h) p38mapk pthwy tivtion in mirogli ells in vivo [7], suggesting tht ERK pthwys medite rther deth thn survivl signling following A exposure. The possible differenes observed in our studies n be due to the different exposure time, timing of mesurements fter A pplition, A onentrtion, speies nd the different signls mesured (phosphoryltion vs. inhibitor expression). Furthermore, we ould not detet delyed, inresed phosphoryltion of p42/44 or p38mapks 2 weeks fter A injetion in the NBM (dt not shown). On the other hnd, our results nd findings from other groups might indite tht A -indued signling pthwy tivtion n lso be time dependent; probbly A indued rpid or delyed tivtion of these signling pthwys in our study, before or beyond our DIGE experiment (less or more thn 24 h). Supporting this ltter hypothesis, we found deresed lbindin (CB) expression fter A injetion. By lowering intrellulr C 2+ buffer pity nd inresing C 2+ onentrtion, deresed lbindin expression ould led to inresed MAPK tivtion in lter time point, nd, ultimtely, ell deth [72, 73]. However, we found no ololiztion of CB nd ChAT signls in the NBM-SI; therefore, A seems to regulte CB expression in other types of neurons or in gli ells. As signifint proportions of the CB-positive ells re likely the ortilly projeting, possibly glutmtergi NBM neurons [52], inresed intrellulr C 2+ onentrtion due to the loss of CB might indue exitotoxiity in the ortex, nd ontribute to holinergi fiber loss nd fiber-loss-indued dying bk mehnism ( fig. 3 ). On the other hnd, E2 prevented RKIP upregultion nd lbindin downregultion nd further deresed PP2A expression, deresing MAPK pthwy inhibition. E2 lso ttenuted lbindin-positive ell loss from the NBM-SI (online suppl. fig. 1). Previously, we demonstrted tht rpid tion of E2 in vivo involves MAPK signling in the NBM-SI in vivo [4], nd link hs been shown to exist between the neuroprotetive effet of E2 nd MAPK tivtion [57]. Altogether, A lters respirtion, metbolism nd signling systems of NBM-SI neurons, nd these dditive effets might ll onverge on deth pthwys [74], but n be redued by E2 pretretment. Interestingly, injetion of A into the NBM-SI indued protein hnges lso in the ortil projetion re. We found upregultion of sepipterin redutse, n enzyme essentil for tetrhydrobiopterin (oftor for ll NOS isoforms), nd dimethylrginine dimethylminohydrolse 1, protein tht inreses NO prodution ( fig. 3 b). However, E2 prevented A -dependent upregultion of both enzymes in the SSCTX. E2 Redues A -Indued Neuron Loss in the NBM Neuroendorinology 13

14 A indued the upregultion of MAP kinse kinse 1 (MEK1) in the SSCTX nd in this wy ontrry to the findings in the NBM A my tivte ERK1/2, p38 or JNK pthwys. Although MEK-tivted pthwys re often ssoited with survivl nd neuroprotetion even in in vivo models [75], A lso tivtes the MAPK sde [76] induing pthologi phosphoryltion of ytoskeletl proteins. We injeted the A in the NBM, nd we found ERK pthwy upregultion just in the projetion re, but not in the NBM. These results suggest tht A - indued signling pthwy tivtion is probbly regulted with different timing in the two res. A might indue first rpid tivtion in the site of injetion (NBM, s suggested in Young et l. [71] ), then derese signling in the NMB but inrese signling in the projetion re, SSCTX (24 h). In ddition, inhibition or permnent tivtion of the sme pthwy might indue the sme response [77, 78]. However, E2 prevented the hnge in MEK1 expression, nd deresed MEK2 level. Moreover, E2 lmost doubled the expression of protein phosphtse 1 (PP1) nd PP2A, proteins responsible mong others for intivtion of some MAPK trgets. Similr to our dt from the ortex, Vlles et l. [2] found tht A indued upregultion of the p38mapk pthwy, wheres this tivtion ws prevented by E2 pretretment. In ddition, E2 ws shown to indue phosphtses to exert neuroprotetive effet in vitro [79]. Therefore, under these onditions, E2 ws ble to reverse the proposed A - indued kinse overtivtion in the SSCTX ( fig. 3 b), nd probbly prtly vi prevention of ortil fibers nd inhibiting dying bk proess, E2 proteted holinergi ell bodies in the NBM-SI. It is well known tht BFC neurons ply n importnt role in lerning nd memory formtion nd tht E2 depletion is ssoited with the ognitive deline observed in AD [24]. In the present study, we demonstrted tht E2 is ble to redue A -indued dmge in the NBM-SI. We found severl ellulr proesses inluding regultion of mitohondril enzymes nd signling pthwys tht ould explin extrellulr A toxiity nd E2 protetion. Colletively, our results demonstrte tht in respet to A, multiple ftors onverge upon pthwys of both A -medited holinergi neurodegenertion nd E2-medited protetion. Aknowledgments We re grteful to Péter Btáry for the ssistne in sttistil nlysis. We thnk Márt Zrándi (Deprtment of Medil Chemistry, University of Szeged) for generously providing the myloid- peptide. This work ws supported by the Regionl Center of Exellene Neurobiologil Center of Exellene in Southern Hungry (RET-DNK to G.D.J., B.P. nd T.J.), by OTKA (68464, 8195 to J.K.), by ETT (to I.M.A.), by the Hungrin Ntionl Offie of Reserh nd Tehnology (NANOAMI KFKT , OMFB-38/26 to I.M.K.) nd by the Deutshe Forshungsgemeinshft through the DFG Reserh Center for Moleulr Physiology of the Brin (CMPB, to E.M.S.). We thnk András Czurkó nd Zsolt Dtki for the helpful disussion, nd Cthy Ludwig for ritil reding of the mnusript. Author Contributions E.M.S., G.D.J. nd J.K. designed the study. E.M.S. performed the in vivo experiments, tissue stining, DIGE nd dt nlysis. K.A.K. performed DIGE nlysis. J.K. performed nd nlyzed AFM dt, Ü.M. nd M.S.Z.K. performed ThioflvinT nd AFM mesurements, B.P. provided the myloid peptide, nd A.C. nd T.J. identified the proteins. M.P. prepred the miropunhes nd G.M. nd I.M.A. prepred histohemil stining. E.M.S. nd G.D.J. wrote the mnusript. Dislosure Sttement The uthors hve no onflits of interest to dislose. Referenes 1 Wimo A, Winbld B, Aguero-Torres H, von Struss E: The mgnitude of dementi ourrene in the world. Alzheimer Dis Asso Disord 23; 17: Mttson MP: Pthwys towrds nd wy from Alzheimer s disese. Nture 24; 43: Shliebs R, Arendt T: The signifine of the holinergi system in the brin during ging nd in Alzheimer s disese. J Neurl Trnsm 26; 113: Mufson EJ, Counts SE, Perez SE, Ginsberg SD: Cholinergi system during the progression of Alzheimer s disese: therpeuti implitions. Expert Rev Neurother 28; 8: Wng HY, Lee DHS, Dvis CB, Shnk RP: Amyloid peptide bet(1 42) binds seletively nd with piomolr ffinity to lph 7 niotini etylholine reeptors. J Neurohem 2; 75: Hernndez CM, Kyed R, Zheng H, Swett JD, Dineley KT: Loss of lph 7 niotini reeptors enhnes bet-myloid oligomer umultion, exerbting erly-stge ognitive deline nd septohippompl pthology in mouse model of Alzheimer s disese. J Neurosi 21; 3: Dineley KT: Bet-myloid peptide niotini etylholine reeptor intertion: the two fes of helth nd disese. Front Biosi 27; 12: Neuroendorinology Szegő et l.