Cyclooxygenase-2 inhibition improves amyloid-b-mediated suppression of memory and synaptic plasticity

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1 doi:1.193/rin/wn8 Brin (28), 131,651^664 Cylooxygense-2 inhiition improves myloid--medited suppression of memory nd synpti plstiity Lind A. Kotilinek, 1, * Mrus A. Westermn, 1, *QinwenWng, 2, *,y Kimerly Pnizzon, 3 Giselle P. Lim, 3 Agnes Simonyi, 5 Sylvin Lesne, 1 Agnieszk Flinsk, 4 Lind H. Younkin, 6 Steven G. Younkin, 6 Mihel Rown, 2 Jmes Clery, 7 Roi Ann Wllis, 3 GreY. Sun, 5 Greg Cole, 3 Slly Frutshy, 3 Roger Anwyl 2 nd Kren H. Ashe 1 1 Deprtment of Neurology, University of Minnesot, Minnepolis, MN 55455, USA, 2 Deprtment of Physiology, Trinity College, Dulin, Repuli of Irelnd, 3 Deprtments of Mediine nd Neurology, UCLA &GLAS, GRECC, VA Hospitl, Sepulved, CA 91343, USA, 4 Neurosiene Deprtment, University of Wles College of Crdiff, Crdiff CF1 3US, UK, 5 Biohemistry Deprtment, University of Missouri, Columi, MO 65212, 6 Myo Clini Jksonville, Jksonville FL nd 7 GRECC, Minnepolis VA Hospitl, Minnepolis, MN 55417, USA *These uthors ontriuted eqully to this work. y Present Address: Deprtment of Physiology, Medil Shool, Ningo University, Ningo , Chin Correspondene to: Kren H. Ashe, MD, PhD, Deprtment of Neurology, MMC 295, 42 Delwre Street SE, University of Minnesot, Minnepolis, MN 55455, USA E-mil: hsio5@umn.edu Non-steroidl nti-inflmmtory gents (NSAIDs) re ssoited with mrked redution in the risk of developing Alzheimer s disese, form of dementi hrterized y the umultion of myloid plques ontining the myloid- protein (A). Studies of the effets of NSAIDs upon the inflmmtory response surrounding myloid plques nd upon the genertion of A from the myloid preursor protein (APP) hve led to two proposed mehnisms y whih NSAIDs my protet ginst Alzheimer s disese: one, the seletive lowering of A42 y suset of NSAIDs; nd two, the redution of inflmmtion. Although Alzheimer s disese is disorder of rin nd synpti funtion, the effets of NSAIDs on A-medited suppression of synpti plstiity nd memory funtion hve never een reported. We therefore investigted how three different NSAIDs, hosen for their distint effets on A42 prodution nd the inhiition of the ylooxygense (COX) isoenzymes, COX-1 nd COX-2, ffet memory funtion nd synpti plstiity. By fousing upon rin nd synpse funtion, we mde novel oservtions out the effets of NSAIDs on A-medited neurl proesses. Here we report tht the seletive inhiition of COX-2, ut not COX-1, utely prevented the suppression of hippompl long-term plstiity (LTP) y A. The non-seletive NSAIDs, iuprofen nd nproxen, nd seletive COX-2 inhiitor, MF-triyli, eh restored memory funtion in Tg2576 mie over-expressing APP, nd lso loked A-medited inhiition of LTP. There ws no dvntge of iuprofen, seletive A42-lowering gent (SALA), over the non-salas, nproxen nd MF-triyli.The enefiil effets on memory did not depend upon lowered levels of A42 or the inflmmtory ytokines, tumour nerosis ftor (TNF-) nd interleukin 1 (IL-1). Intriguingly, improved memory funtion ws inversely relted to prostglndin E2 (PGE2) levels. Conversely, exogenous PGE2 prevented the restortive effets of COX-2 inhiitors on LTP. The dt indite tht the inhiition of COX-2 loks A-medited suppression of LTP nd memory funtion, nd tht this lok ours independently of redutions in A42 or dereses in inflmmtion. The results led us to propose third possile mehnism y whih NSAIDs my protet ginst Alzheimer s disese, involving the lokde of COX-2-medited PGE2 response t synpses. Downloded from t Trinity College Lirry, Dulin on June 18, 21 Keywords: NSAIDs; inflmmtion; trnsgeni; memory; synpti plstiity ß The Author (28). Pulished y Oxford University Press on ehlf ofthe Gurntors of Brin. Allrights reserved. For Permissions, plese emil: journls.permissions@oxfordjournls.org

2 652 Brin (28), 131,651^664 L.A.Kotilineket l. Arevitions: APP = myloid preursor protein; A = myloid- protein; COX-1 nd COX-2 = ylooxygense isoenzymes; IL-1 = interleukin 1; LTP = long-term plstiity; MPS = men proe sore; NSAIDs = Non-steroidl nti-inflmmtory gents; PGE2 = prostglndin E2; SALA = seletive A42-lowering gent; TNF- = tumour nerosis ftor. Reeived My 17, 27. Revised Otoer 31, 27. Aepted Jnury 1, 28 Introdution Non-steroidl nti-inflmmtory gents (NSAIDs) hve een ssoited with up to n 8% redution in the inidene of Alzheimer s disese (in t Veld et l., 21), ut the underlying protetive mehnism is unknown. Given the potentil enefits of NSAIDs in individuls t risk for Alzheimer s disese, it is importnt to understnd the mehnism y whih this lss of drugs my protet memory nd synpti plstiity using in vitro nd in vivo models. More thn 2 trgets of NSAIDs hve een identified (Tegeder et l., 21; Weggen et l., 21). The mjor NSAID trgets involved in inflmmtory sdes nd myloid preursor protein (APP) proessing re shown in Fig. 1. COX-1 nd COX-2 re the prinipl trgets of NSAIDS t low doses etween 1 nd 1 mm (Tegeder et l., 21). COX-1 nd COX-2 tlyse the formtion of prostglndins, inluding prostglndin E2 (PGE2), from rhdoni id. The tivtion or suppression of other trgets, inluding peroxisome prolifertor-tivted reeptors PPARs nd nuler ftor-kb, generlly requires higher onentrtions (45 mm) of NSAIDs (Tegeder et l., 21). Non-seletive NSAIDs inhiit oth COX-1 nd COX-2. Seletive inhiitors of COX-2 re lled oxis. In Alzheimer s disese, miroglil ells expressing COX-1 surround A deposits (Hoozemns et l., 21), while COX-2 umultes in neurons (Psinetti nd Aisen, 1998; Ho et l., 21; Hoozemns et l., 21). The protetive effets of NSAIDs hve een sried to their ntiinflmmtory properties (MGeer, 2; vn Gool et l., 23), ut epidemiologil dt supporting the effiy of low-dose (elow nti-inflmmtory doses) NSAIDs rgue ginst the nti-inflmmtion effet (Broe et l., 2). A suset of NSAIDs, inluding iuprofen ut not nproxen or the oxis, lowers prodution of the highly myloidogeni 42-residue form of myloid- protein (A), A42, y modulting g-seretse tivity (Weggen et l., 21; Eriksen et l., 23; Zhou et l., 23). This COXindependent tivity hs een proposed s n lterntive explntion to nti-inflmmtion s the mehnism y whih NSAIDs exert their neuroprotetive effets (Weggen et l., 21). In most tissues, COX-1 is onstitutively expressed, while COX-2 is indued in response to injury. However, neurons differ euse they express COX-2 during oth physiologil nd pthologil proesses. The evidene for physiologil role of COX-2 is derived from the oservtions tht there re high sl levels of COX-2 in the hippompus nd ererl ortex, tht tivtion of NMDA reeptors trnsiently inreses COX-2 expression, whih is onentrted in dendriti spines (Ymgt et l., 1993; Kufmnn et l., 1996), nd tht high-dose oxis interfere with memory onsolidtion (Tether et l., 22). Support for pthologil role of COX-2 omes from dt showing tht COX-2 expression in neurons is indued y A, glutmte nd inflmmtory ytokines (Fig. 1) (Too et l., 1997; Psinetti nd Aisen, 1998; Bzn, 21), nd tht PGE2 levels re inresed in Alzheimer s disese (Montine et l., 1999). () () Possile Trgets of NSAIDs in Prevention of AD Inhiition t doses of 1-1µM Ativtion or suppression generlly t doses >5µM Arhidoni id Cylooxygenses1 & 2 ( ) Lipoxygenses( ) Prostnoids Leukotrienes NF-kB ( ) PPAR-g ( ) PPAR- ( ) Aß Aβ Oxidtive Inflmmtory stress ytokines Glutmte COX-2 ALZHEIMER S DISEASE Amyloid preursor protein β-seretse g-seretse ( ) Aβ42 ( ) Fig. 1 Possile trgets of NSAIDS in the prevention of Alzheimer s disese nd proposed pthophysiology of memory loss in the Tg2576 mouse model of Alzheimer s disese. () The tivities of enzymes nd ftors (lue nd red lettering) re ffeted y NSAIDs in the diretion indited y the rrow in prentheses or y the delt sign signifying qulittive ltertion in tivity. The ylooxygenses COX-1 nd COX-2 re mjor trgets of NSAIDs euse their inhiition n e hieved t doses etween 1 nd 1 mm. The tivtion or suppression of other trgets, inluding peroxisome prolifertors-tivted reeptors (PPARs) nd nuler ftor-kb (NF-kB), generlly requires higher onentrtions (45mM) of NSAIDs. Non-seletive NSAIDs inhiit oth COX-1 nd COX-2. The oxis seletively inhiit COX-2. A suset of NSAIDs, inluding iuprofen ut not nproxen or the oxis, lowers prodution of A42, the highly myloidogeni 42-residue form of A, y modulting g-seretse tivity. () In Alzheimer s disese COX-2 is stimulted y A, glutmte nd inflmmtory ytokines, whih in turn re modulted y COX-2. Downloded from t Trinity College Lirry, Dulin on June 18, 21

3 NSAIDs improve A-medited memory loss Brin (28), 131, 651^ APP trnsgeni mie develop little or no loss of neurons or synpses (Irizrry et l., 1997, ), yet nevertheless exhiit ge-relted memory loss (Hsio et l., 1996; Chen et l., 2; Westermn et l., 22), whih is rpidly reversile (Dodrt et l., 22; Kotilinek et l., 22). A mehnisti explntion for these oservtions hs een provided y the oservtion tht oligomeri ssemlies of A re oth neessry nd suffiient for A to disrupt ognition without induing permnent neurologil defiits (Clery et l., 25), nd tht speifi 56-kD solule ssemly of A, lled A 56, ppers to impir memory in the well-hrterized Tg2576 model of Alzheimer s disese lking neurodegenertion (Lesné et l., 26). Solule oligomers of A lso inhiit hippompl long-lsting synpti plstiity (Wlsh et l., 22; Wng et l., 22, 24). These oservtions suggest tht APP trnsgeni mie represent prodroml or ltent stge of Alzheimer s disese rther thn the tul disese itself (Zndi nd Breitner, 21; Ashe, 25; Lesné et l., 26). Tg2576 mie my therefore e good model in whih to exmine prophylti mehnisms. More thn 1 studies of NSAIDs in trnsgeni APP mie hve doumented their effets on myloid lod nd inflmmtion (reviewed in MGeer nd MGeer, 27), ut none to dte hve reported the effets of NSAIDs on A-medited disruption of synpti plstiity nd memory. Therefore, we used well-estlished in vitro nd in vivo model systems to exmine the effets of NSAIDs on A-medited inhiition of synpti plstiity nd memory, mesuring sptil referene memory in Tg2576 mie nd LTP in rt hippompl slies exposed to solule A oligomers (Wng et l., 24). Our oservtions indite tht NSAIDs shring the ommon property of loking COX-2 meliorte A-indued neuronl dysfuntion, independently of n nti-inflmmtory effet or redution in the levels of A, inluding A42 nd A 56. Importntly, improved memory funtion orrelted with deresed PGE2 levels. Moreover, the ility of COX-2 inhiitors to resue A-medited inhiition of LTP ws loked y the ddition of exogenous PGE2. Beuse COX-2 in rodent rins is onentrted in dendriti spines (Ymgt et l., 1993; Kufmnn et l., 1996), our results suggest role for PGE2 signlling in dendriti spines in A-medited synpti nd memory dysfuntion. Mteril nd Methods Long-term potentition studies Trnsverse slies (35 mm) of the rt (mles, ge 3 4 weeks, weight 4 8 g) hippompus were pled in oxygented medium t room temperture for 1 h, then ontinuously superfused in the reording hmer t 3 32 C. The ontrol medium ontined: (mm) NCl, 12; KCl 2.5, NH2P4, 1.25; NHC3 26; MgS4, 2.; CCl2, 2.; d-gluose 1. All solutions ontined 1 mm pirotoxin (Sigm, St Louis, MO) to lok GABA-medited tivity. Drugs used were NS-398, piroxim (oth from Toris Cookson Ltd, Bristol, UK), MF triyli (Merk, Whitehouse Sttion, NJ), iuprofen, nproxen nd PGE2 (from Cymn Chemil, Ann Aror, MI). NS-398 nd piroxim were dissolved in DMSO, with mximum finl onentrtion of.1% DMSO. MF triyli ws dissolved in etone, with finl onentrtion of etone of.375%. Iuprofen, nproxen nd PGE2 were dissolved in rtifiil CSF medi. Syntheti A 1-42 (Bhem UK Ltd, St Helens, UK) ws prepred s stok solution of 5 mm in mmonium hydroxide (.1%), stored t 2 C nd dded immeditely prior to eh experiment. Presynpti stimultion ws pplied to the medil perfornt pthwy of the dentte gyrus using ipolr insulted tungsten wire eletrode, nd field exittory postsynpti potentils (EPSPs) were reorded with glss miroeletrode t ontrol test frequeny of.33 Hz from the middle one-third of the moleulr lyer of the dentte gyrus. An input output urve (fferent stimulus intensity versus EPSP mplitude) ws plotted t the test frequeny. The mplitude of the test EPSP ws djusted to one-third of mximum (1.2 mv). Long-term plstiity (LTP) ws evoked y high-frequeny stimultion (HFS) onsisting of eight trins, eh of eight stimuli t 2 Hz, intertrin intervl 2 ms, with the stimultion voltge inresed during the HFS so s to eliit n initil EPSP of the trin of doule the norml test EPSP mplitude. The mesurements of LTP re t 6 min post-hfs. NSAID tretment Tg2576 mie were in hyrid C57B6/SJL (B6SJL) (Hsio et l., 1996) or ongeni 129S6 FVB F1 (129FVBF1) kground. We used B6SJL in the prophylti study nd 129FVBF1 in the restortion study. Groups were ounterlned y gender nd litter. Drugs were dministered orlly y supplementing sl feeding how. Iuprofen (375 p.p.m.; Sigm, St Louis, MO) nd nproxen (375 p.p.m.; BIOMOL, Plymouth Meeting, Pennsylvni) were prepred in sl how NIH-31 y Hrln Tekld (Mdison, WI) nd MF triyli (13 p.p.m.; Merk, Whitehouse Sttion, NJ) ws prepred in LDiet 51 y Merk. The phrmokinetis nd seletivity of MF triyli for COX-2 over COX-1 is lmost identil to rofeoxi (Oshim et l., 1996). Behviourl tests Sptil referene memory ws mesured using the Morris wtermze (Westermn et l., 22). Sine 129FVBF1 mie lern more rpidly thn B6SJL mie, testing ws tilored for eh kground strin (Westermn et l., 22). B6SJL mie reeived visile pltform trining for 3 dys, eight trils per dy, followed y hidden pltform trining for 9 dys, four trils per dy. Three proe trils were performed 2 h fter 12, 24 nd 36 trining trils, nd the verge trget qudrnt oupny for the three proe trils (men proe sore), ws lulted. Visile pltform trining of 11.6-month nd 12.5-month 129FVBF1 mie ws done for 2 dys, six trils per dy or 3 dys, 6 trils per dy respetively, followed y hidden pltform trining for 6 dys, four trils per dy. In 11.6-month 129FVBF1 mie, four proe trils were performed 2 h fter 4, 8, 16 nd 24 trining trils. In 12.5-month 129FVBF1 mie, five proe trils were performed 2 h fter 4, 8, 12, 16 nd 24 trining trils. The men proe sore (MPS) ws the men trget qudrnt oupny of proes otined etween the 8th nd 17th trils. Proe trils lsted 6 s, ut MPSs in 129FVBF1 Downloded from t Trinity College Lirry, Dulin on June 18, 21

4 654 Brin (28), 131,651^664 L.A.Kotilineket l. mie were lulted using the first 3 s euse they exhiited extintion. Sptil working memory ws tested using fored-hoie lterntion in T-mze, desried previously (Chpmn et l., 1999). Rewrd ws 25% surose in tp wter. The test onsisted of 4 dys of dpttion to the T-mze (5 min/dy) nd 1 onseutive dys of quisition with six pirs of trils per dy with fored hoies pseudo-rndomly ssigned to left nd right. Mie with neophoi mnifested y immoility were exluded. IL-1 nd tumour nerosis ftor- (TNF-) Interleukin 1 (IL-1) ws mesured y ELISA (Lim et l., 2). (TNF-) ws mesured y ELISA using monolonl nti-mouse TNF- pture ntiody (MM-35C; Endogen, Wourn, MA). COX-2 In situ hyridiztion ws rried out s desried for COX-2 mrna in rt rin tissue (Simonyi et l., 22), exept tht sgittl insted of oronl setions were used. PGE2 Forerin (minus hippompus) tissue ws weighed, homogenized nd sonited, nd etone dded. Smples were entrifuged t 15 g for 1 min nd the superntnt dried y vuum entrifugtion. The dried smples were reonstituted in uffer, nd PGE-2 levels were determined y EIA (Cymn Chemil) ording to mnufturer s methods. Eh smple ws tested in duplite nd levels represented s pg PGE-2/mg tissue. Iuprofen,nproxenndMFtriyli Plsm smples were mixed with two volumes of etonitrile nd mouse rins were homogenized in five equivlents of wter nd entrifuged. Quntittion of the ompounds in extrted superntnts ws rried out y HPLC nd tndem mss spetrometri (LC/MS/MS) detetion. Tndem mss spetrometri (MS/MS) detetion ws rried out in negtive ion mode y multiple retion monitoring (iuprofen t m/z 25!161., nproxen t m/z 229.! 17. nd MF triyli t m/z 349.! 32.7). A A4 nd A42 were mesured y ELISA in SDS nd formi id extrts of forerin (minus hippompus) tissue, using the 316 polylonl pture ntiody (Kwryshi et l., 21). A 56 A 56 ws mesured in memrne-enrihed extrts of hemirins following four-step extrtion proedure, y immunolotting using the 6E1 monolonl ntiody (Lesné et l., 26). Sttistil nlysis ANOVA nd Student s t-tests were used. Results Effets of COX inhiition on A-indued inhiition of LTP To ddress the importne of synpti COX-2 in mediting the enefiil effets of NSAIDS on memory, we exmined the involvement of COX-2 in the indution of LTP in rt hippompl slies exposed to solule, syntheti A42, s desried previously (Wng et l., 24). A42 prevented the indution of LTP t the medil perfornt pth to dentte gyrus grnule ell, LTP mesuring 161 4% in ontrol nd 17 5% in the presene of A42 (n =5, P5.1) (Fig. 2). The seletive COX-2 inhiitors MF triyli nd NS398 prtilly prevented the inhiition of LTP y A42, LTP mesuring 134 8% (n =5, P5.1) nd 143 3% (n =5, P5.1) in the presene of A plus MF triyli nd NS-398, respetively (Fig. 2 nd ). In ontrst, the seletive COX-1 inhiitor piroxim filed to restore LTP disrupted y A, LTP mesuring 15 7% in the presene of A plus piroxim (n =5, P4.5) (Fig. 2d). Neither the COX-2 inhiitors MF triyli nd NS398 nor the COX-1 inhiitor piroxim ltered LTP indution in the sene of A, LTP mesuring 152 5%, 163 4% nd 157 7% in the presene of MF triyli, NS398 nd piroxim, respetively (n =5, P4.5) (Fig. 2 d). Non-seletive NSAIDs lso lrgely prevented the inhiition of LTP y A42, LTP mesuring 152 6% (n = 6, P5.1) nd 153 6% (n =6, P5.1) in the presene of A plus iuprofen nd nproxen, respetively (Supplementry Fig. 1), ut neither drug ltered LTP indution in the sene of A, LTP mesuring 163 7% nd 167 6%, respetively. Thus the inhiition of synpti COX-2 tivity ws suffiient to restore longlsting potentition whih hd een disrupted y solule, syntheti A42. Effets of COX inhiition on sptil memory intg2576 mie To sertin the effets of COX inhiition on the restortion of memory in Tg2576 mie (Hsio et l., 1996), we tested two non-seletive COX inhiitors, iuprofen nd nproxen, nd one seletive COX-2 inhiitor, MF triyli (Oshim et l., 1996), nd dministered dosges equivlent to low to moderte doses in humns (e.g mg iuprofen per dy), sed upon serum level omprisons (Tle 1). Brin levels of these drugs were 1 3 mm (Tle 1). At these onentrtions, their priniple ommon tivity is to inhiit COX-2, thus exluding most other potentil trgets of NSAIDs (Tegeder et l., 21). Memory loss in Tg2576 mie egins t 6 months of ge (Fig. 3) (Westermn et l., 22). NSAIDs were strted t 11.5-months nd mie were treted for 4 weeks efore ehviourl testing ws egun (Fig. 3). Using the Morris wter-mze tilored to Tg2576 mie, we defined n index of sptil referene memory lled the men proe sore Downloded from t Trinity College Lirry, Dulin on June 18, 21

5 NSAIDs improve A-medited memory loss Brin (28), 131, 651^ () () 1mV 1ms 1mV 1ms 25 A et1-42 A-et 25 MF-triyli EPSP (% ontrol) EPSP (% ontrol) () HFS ontrol A-et Time (min) Time (min) (d) HFS NS398 A A-et mV 1ms NS398 NS398+A-et EPSP (% ontrol) EPSP (% ontrol) HFS MF-triyli MF-triyli+A et Time (min) HFS A et1-42 A-et piroxim piroxim+a-et Time (min) piroxim A A-et mV 1ms Downloded from t Trinity College Lirry, Dulin on June 18, 21 Fig. 2 Seletive COX-2, ut not COX-1, inhiitors prevent the inhiition of LTP indution y syntheti, solule A42. () LTP indued y single rief high-frequeny stimultion (HFS) (losed irles) nd LTP indution in the presene of syntheti, solule A42 (5 nm), pplied 4 min prior to HFS (open irles). LTP in the presene of syntheti, solule A42 ws signifintly redued from ontrol. () The COX-2 inhiitor MF triyli (3 mm), pplied 6 min prior to HFS, does not inhiit LTP indution (losed irles) ut prevents the A-medited inhiition of LTP indution (open irles). () The COX-2 inhiitor NS-398 (2 mm), pplied 6 min prior to HFS, does not inhiit LTP indution (losed irles) ut prevents the A-medited inhiition of LTP indution (open irles). (d) The COX-1 inhiitor piroxim (1 mm), pplied 6 min prior to HFS, does not inhiit LTP indution (losed irles) nd lso does not prevent the A-medited inhiition of LTP indution (open irles). The tres, nd re field EPSPs t the times indited y, nd on the grphs, with the top set of tres orresponding to the losed irles nd the lower set of tres to the open irles.

6 656 Brin (28), 131,651^664 L.A.Kotilineket l. Tle 1 Drug levels in NSAID-treted mie Drug 3 months old 13 months old Brin (mm) Serum (mm) Brin/Serum Rtio (%) Serum (mm) Iuprofen (12) (12) 2.87%.17 (12) (1) Nproxen (6) (6) 2.35%.36 (6) (1) MF triyli (6) (6) 4.14% 2.39 (6) (1) Iuprofen, nproxen nd MF triyli re present in the rin nd serum of g2576 mie following orl NSAID tretment for 3 dys (3 months old) nd 4 dys (13 months old) respetively. Dt re presented s men SD (no. of mie). (MPS) (Westermn et l., 22). Anlysis of MPSs in treted mie reveled signifint tretment group y trnsgene sttus intertion [F(3,135) = 3.1, P =.3], inditing tht tretment signifintly ltered ehviourl outome only in trnsgene positive (Tg + ) mie (Fig. 3 nd ), in whih there ws signifint min effet of tretment [F(3,68) = 3.6, P =.2]. Tg + mie fed iuprofen, nproxen or MF triyli showed signifintly higher MPSs thn mie fed ontrol how (P =.4, P =.6 nd P =.36, respetively). NSAIDs did not impir memory signifintly in non-trnsgeni (Tg ) mie (Fig. 3). In mie fed ontrol-how, ut not NSAID-hows, there ws signifint min effet of trnsgene sttus (P5.1), showing tht NSAIDs eliminted the distintion etween Tg + nd Tg littermtes. To determine if improved performne in the wter mze ould e ttriuted to potentil nlgesi effets of NSAIDs, we ompred swim speeds nd found no signifint min effet of tretment (dt not shown). These results indite tht loking COX-2 ws suffiient to restore memory in Tg2576 mie. We lso exmined the effets of prophylti iuprofen dministrtion. We initited iuprofen t 4.5 months of ge, nd tested sptil working memory using fored hoie lterntion in T-mze, desried previously (Chpmn et l., 1999), eginning t 8.5 months nd sptil referene memory strting t 9.5 months (Fig. 4). Iuprofen-treted Tg + mie mde signifintly higher perentge of orret hoies during the hoie tril in the T-mze ompred to Tg + mie on ontrol diet Similrly, iuprofen-treted Tg + mie hd signifintly higher MPSs in the wter mze thn Tg + mie on ontrol diet (Fig. 4), performing t levels similr to Tg littermtes shown previously (Westermn et l., 22). These results show tht iuprofen prevented loss of sptil working nd referene memory (Fig. 4). EffetsofNSAIDsoninflmmtory ytokines Beuse NSAIDs hve nti-inflmmtory properties, we exmined the orrespondene etween memory nd neurotoxi inflmmtion. At 8.5 to 9 months, the erliest ge in whih we found enefiil effets of NSAIDs, the hippompus of Tg2576 mie is virtully devoid of plques nd onsequently there is no plque-ssoited miroglil response (Frutshy et l., 1998). A-stimulted mirogli hve een shown to produe TNF- oth in ultured ells nd in Tg2576 mie (Tn et l., 1999). We therefore mesured hippompl TNF- nd found no signifint elevtions in 1.5-month Tg + mie (Fig. 5), onsistent with the miniml mount of myloid deposition t this ge. In ontrst, TNF- levels were elevted y ftor of 2-fold in 13-month-old Tg + mie, onsistent with previous studies (Tn et l., 1999), ut TNF- expression ws not redued y NSAIDs in 13-month-old Tg + mie (dt not shown). To extend our investigtions, we mesured levels of nother pro-inflmmtory ytokine, IL-1, whih hs een shown to e elevted in Alzheimer s disese nd to inhiit long-lsting synpti plstiity in the hippompus, nd hs therefore een implited in memory loss in Alzheimer s disese (Celos et l., 1994; Murry nd Lynh, 1998). In 16-month Tg2576 mie, we previously demonstrted n elevtion in hippompl IL-1 ssoited with the presene of undnt plques (Lim et l., 2). In the urrent study, we found signifint inreses in hippompl IL-1 in 1.5-month Tg + mie reltive to Tg littermtes (P5.1) (Fig. 5). In ddition, we ompred levels of hippompl IL-1 in 1.5-month nd 13-month Tg + mie to determine whether IL-1 expression ws upregulted in n ge-relted fshion. As expeted, we oserved tht IL-1 levels were inresed with geing in Tg + mie (P5.1) (Fig. 5). Treting mie with Iuprofen suppressed the rise in hippompl IL-1 in 1.5-month-old Tg + mie (Fig. 5), s reported previously (Lim et l., 2). These results indite tht elevtions in IL-1 preeded signifint plque deposition, nd suggested tht IL-1 my ontriute to memory loss in Tg2576 mie. To investigte whether the reovery of ognitive funtion in NSAID-treted Tg2576 mie ws relted to the modultion of IL-1 levels, we exmined 13-month Tg2576 mie treted with NSAIDs for 6 weeks. If elevted levels of IL-1 were neessry for impiring ognitive funtion, then we would expet the restortion of memory to e ompnied y redutions of IL-1 to levels lose to those found in non-trnsgeni mie. Despite modest lowering of IL-1 in NSAID-treted Tg + mie, IL-1 levels remined 42.5-fold higher thn in non-trnsgeni mie (P5.1) (Fig. 5d). Importntly, IL-1 levels in 13-month, NSAID-treted Tg + mie with intt memory were 41.7 times higher thn in Downloded from t Trinity College Lirry, Dulin on June 18, 21

7 NSAIDs improve A-medited memory loss Brin (28), 131, 651^ () Onset of memory loss & pperne of Aβ*56 in Tg2576 mie () Onset of memory loss & pperne of Aβ*56inTg2576 mie () () % Time MPS Trining trils # Iuprofen Nproxen MF triyli Test 1 Test Test 1 Test 2 ** ** * Trnsgene sttus Tretment group + + Iuprofen + Nproxen + MF triyli Iuprofen Nproxen MF triyli No Tretment n Trnsgene Age (months) Amyloid plques Aβ*56 Sptil memory Age (months) Fig. 3 Reversl of memory defiits nd restortion of sptil memory y dministrtion of the onventionl NSAIDs, iuprofen nd nproxen, nd the seletive COX-2 inhiitor, MF triyli, in Tg2576 mie. () Experimentl design. Wedges nd retngles ove the timeline qulittively represent myloid plques (stippled), A 56 (drk grey) nd memory ility (lk) in Tg2576 mie s funtion of ge. Mie52 months were too young to test. Brs underneth the timeline indite the durtion of tretment. Arrows indite the initition of ehviourl tests. To void possile retest effets relted to the short inter-test intervl, Tests 1 nd 2 were performed on different groups of mie. () Aquisition of memory in NSAID-treted nd ontrol Tg2576 mie nd non-trnsgeni littermtes. Dt re the men trget qudrnt oupny during proe trils s funtion of numer of trining trils. () The men proe sore (MPS) in NSAID-treted nd ontrol Tg2576 mie nd non-trnsgeni littermtes. MPS is the men trget qudrnt oupny in proes performed etween the eighth nd 17th trils. Rndom swimming in ll three proe trils would yield n MPS of 25. Ages t initition of testing re indited. Dt re the men SEM of the MPS for eh mouse ( P5.5; P5.1, ompred to ge-mthed Tg2576 mie on ontrol diet; # P5.5 ompred to ge-mthed nontrnsgeni mie). Visile pltform pth lengths during the finl three trining trils were equivlent for month nd month Tg2576 nd non-trnsgeni littermtes (dt not shown). untreted 1.5-month Tg + mie with impired memory (P5.1) (Fig. 5e). A similr reltionship ws oserved for TNF- (dt not shown). These studies dissoite IL-1 nd TNF- from memory loss, nd suggest tht the mehnism y whih NSAIDs re le to restore memory () 9 % Corret Amyloid plques Aβ*56 Sptil memory Age (months) Iuprofen (16) Iuprofen (16) T-mze Wter-mze ** ** Bloks of two trining dys () 6 funtion in Tg2576 mie is not relted to their ntiinflmmtory properties trgeting TNF- or IL-1. EffetsofNSAIDsonA We then sked whether the enefiil effets of NSAIDs were relted to redutions in A. NSAIDs n stimulte A lerne y tivting mirogli (Jntzen et l., 22), nd suset of NSAIDs n redue A42 prodution (Weggen et l., 21). We found tht the levels of solule (SDS) nd firillr (FA) A4 nd A42 in Tg + mie were not modified y tretment with NSAIDs (Fig. 6). In ddition, oth solule nd firillr A levels remined 41 times higher thn those evluted in Tg + mie 56 months of ge, prior to the onset of memory loss (Kwryshi et l., 21). This result indites tht lthough NSAIDs fully restored ognitive funtion, they filed to redue A levels to those found in young, ognitively intt mie. The sene of effets on A levels ssoited with nproxen nd MF triyli ws onsistent with previous reports tht nproxen nd rofeoxi re inple of lowering A42 in vivo or in vitro (Weggen et l., 21; Eriksen et l., 23; Kukr et l., 25). The filure of iuprofen to redue A ws unexpeted initilly, given previous findings tht iuprofen n seletively redue rin A42 in 3-month Tg2576 mie (Weggen et l., 21; Eriksen et l., 23), ut is onsistent with more reent findings y n independent group showing iuprofen does not redue A levels in plque-free, young Tg2576 mie MPS (16) Iuprofen (16) Fig. 4 Preservtion of sptil memory y hroni dministrtion of iuprofen in Tg2576 mie. () Experimentl design. () Sptil working memory in iuprofen-treted nd ontrol Tg2576 mie, ssessed using thet-mze. Dt re the men SEM of the perentge of orret responses verged over two dys for eh mouse (F = 5.6, P =.2, for tretment effet y multiple-mesures ANOVA, P5.1 y t-test). Numers of mie re in prentheses. () Sptil referene memory in iuprofen-treted nd ontrol Tg2576 mie, ssessed using the Morris wter-mze. Dt re the men SEM of the MPS for eh mouse (F =5.13, P =.3, for tretment effet y ANOVA). * Downloded from t Trinity College Lirry, Dulin on June 18, 21

8 658 Brin (28), 131,651^664 L.A.Kotilineket l. () 6 IL-1 (fg/mg) 4 2 # () TNF (pg/mg) * * # (Lnz et l., 25). To investigte this issue further, we nlysed the effet of our dosge regimen on rin A42 in plque-free 2-month Tg2576 mie treted for 3 dys nd found no effets (Fig. 6). Brin iuprofen levels (Tle 1) were within the rnge of onentrtions reported to redue A42 in Tg2576 mie (Eriksen et l., 23), whih reeived the sme totl mount of iuprofen per dy s the mie in our experiments. The redution of plque urden previously oserved with the sme orl regimen in how (Lim et l., 2; Yn et l., 23) my involve effets of iuprofen tht depend upon the plque lod nd inflmmtory stte in the rin (Henek et l., 2; Morihr et l., 25). Importntly, our results show tht memory funtion ws restored following NSAIDs regrdless of their A42 lowering pilities, sine improved memory ws oserved with nproxen whih hs no predited or oserved effets on lowering A42 (Weggen et l., 21; Kukr et l., 25). Beuse A 56 ppers to impir memory in Tg2576 mie (Lesné et l., 26), we sked whether NSAIDs ltered C IB Tretment + + Trnsgene 1.5 Age (mo.) Age (mo.) (d) IL-1 (fg/mg) () IL-1 (fg/mg) ** **** C C IB Np MF Tretment Trnsgene 13 Age (mo.) * * Age (mo.) (e) 6 IL-1 (fg/mg) 4 2 Non-Tg * * ** Non-Tg Iuprofen Nproxen MFtriyli C IB Np MF Tretment Trnsgene Age (mo.) Impired Intt Memory Fig. 5 TNF- nd IL-1 levels in Tg2576 mie. () TNF- is signifintly elevted in 13-month Tg2576 mie. Dt re the men SEM of hippompl TNF- mesured y ELISA ( # P5.1; P5.1). TNF- is not signifintly redued in 13-monthTg2576 mie treted with NSAIDs (dt not shown). () Age-relted inrese in IL-1 in Tg2576 mie. Dt re the men SEM of hippompl IL-1 mesured y ELISA ( P5.1). () IL-1 is suppressed in 1.5-month Tg2576 mie given iuprofen prophyltilly ( # P5.1). (d) IL-1 remins signifintly elevted in 13-monthTg2576 mie treted with NSAIDs ( P5.1). (C), Iuprofen (IB), Nproxen (Np), MF triyli (MF). (e) Memory-intt 13-month Tg2576 mie treted with NSAIDs showed signifintly higher levels of IL-1 thn memory-impired 1.5-monthTg2576 mie on ontrol diet. ( P5.1; P5.1). A similr reltionship ws oserved for TNF- (dt not shown). Numers of mie denoted inside rs. its onentrtion in the rin. We found no effet of iuprofen tretment on A 56 levels (Fig. 6). The dt indite tht the enefiil effets of NSAIDs on memory ourred independently of inhiiting the prodution of A42 through g-seretse modultion, reduing the umultion of A4, A42 or A 56 or inresing the lerne of A in the rin. COX-2 levels intg2576 mie Sine the eletrophysiologil nd ehviourl dt supported the involvement of COX-2 in impiring neuronl funtion, we sked whether the enefiil effets of COX-2 inhiition depended upon the elevtion of COX-2 in Tg2576 mie. We ompred COX-2 messenger RNA (mrna) y in situ hyridiztion in 4, 8 nd 12-month Tg + nd Tg littermtes, whih permitted semi-quntittive studies in different rin regions. Consistent with mesurements using n mrna protetion ssy (Sung et l., 24), we found no inrese in COX-2 expression in the hippompus or the Downloded from t Trinity College Lirry, Dulin on June 18, 21

9 NSAIDs improve A-medited memory loss Brin (28), 131, 651^ () () happ A*56 CTF 1-mer happ A*56 CTF 1-mer A (pmol/g) C + C Iuprofen Nproxen MF triyli A4 A42 A4 A42 A42 SDS Memrne-enrihed extrts I I I C C C C I I I I C I I C C I I I I C C WB 6E1 Fig. 6 A levels intg2576 mie. () No signifint effets of tretment on A levels in mie treted with NSAIDs from 11.5 to 13 months of ge. Forerin A4 nd A42 levels were mesured y ELISA in SDS nd formi id (FA) frtions. Numers of mie denoted inside rs. () No signifint effets of tretment on A42 levels in 2-month-old mie treted with NSAIDs for 3 dys. Brins were extrted in SDS nd A42 levels were mesured y ELISA. () A 56 levels re not modulted y iuprofen tretment. Tg2576 mie ( + ) nd non-trnsgeni littermtes (^) were treted with iuprofen (I) or ontrol (C) diets from 11.5 to 13 months of ge. Forerin A 56 ws mesured in memrne-enrihed extrts y Western lotting (WB; 1 mg protein/lne) using 6E1 monolonl ntiodies (1:1,). CTF nd monomeri A (1-mer) levels lso showed no signifint ltertions. FA Reltive levels (% of untreted Tg +) () A (pmol/g) 1 happ C A*56 C I I SDS CTF C 1-mer C I I Downloded from t Trinity College Lirry, Dulin on June 18, 21 ererl ortex (Fig. 7), oserving insted non-signifint derese in COX-2 mrna in 8 nd 12-month Tg + mie ompred to Tg littermtes. The lk of hnge in COX-2 is onsistent with other reports in ged Tg2576 mie showing no inrese in prostglndin E2 (PGE2), the mjor oxidtion produt of rhidoni id nd COX-2, nd no inrese in COX-2 mrna in mirorry nlyses (Dikey et l., 23; Quinn et l., 23). The low levels of COX-2 in Tg2576 further support our ssumption tht the mie represent prodroml or ltent phse of Alzheimer s disese, rther thn linilly pprent Alzheimer s disese. Disruption of sptil memory nd synpti plstiity y A is dependent upon COX-2-medited PGE2 signlling Beuse NSAIDs lok the ility of COX-2 to tlyse the formtion of PGE2 from rhdoni id, we lso mesured the levels of PGE2, nd found tht they were lower in Tg + mie ompred to Tg littermtes (Fig. 7). Next, we evluted the effets of NSAIDs on PGE2 levels. While COX inhiitors onsistently redue elevted levels of PGE2 in pthologil sttes, their effets on physiologil

10 66 Brin (28), 131,651^664 L.A.Kotilineket l. () Hippompus (m Ci/g) () PGE2 (pg/mg) Months * Iuprofen Nproxen MF triyli C IB Np MF C IB Np MF Tretment Trnsgene Age (mo.) HIP Non-Tg HIP Tg2576 CTX Non-Tg CTX Tg2576 Fig. 7 COX-2 mrna nd PGE2 levels in Tg2576 mie. () COX-2 expression is not elevted in Tg2576 mie. COX-2 mrna ws mesured y in situ hyridiztion in 4-, 8- nd 12-month Tg2576 mie (n = 1) nd non-trnsgeni littermtes (n = 9). Dt re the men SEM of COX-2 mrna in the CA3 region of the hippompus (HIP) nd frontl ortex (CTX). () PGE2 levels re signifintly lower in 13-month Tg2576 mie. No signifint effet of NSAID tretment on PGE2 levels ws oserved. Dt re men SEM of forerin (minus hippompus) PGE2 mesured y EIA ( P5.1). PGE2 levels re inonsistent, with some reports showing no effets nd others showing dereses (Bik et l., 1999; Klivenyi et l., 24). We found no signifint effet of NSAIDs on overll PGE2 levels in the forerin tissue of Tg2576 mie (Fig. 7). The PGE2 dt ws onsistent with the COX-2 mrna results, nd indite tht the enefiil effets of NSAIDs on memory funtion do not require inreses in COX-2 expression or PGE2 levels. To more rigorously link the inhiition of COX-2 tivity with the reovery of memory, we sertined the reltionship etween memory nd levels of PGE2, TNF-, IL-1, A4 nd A42 in Tg2576 mie treted with NSAIDs. We divided Tg + mie into three evenly sized ins sed upon performne in the wter mze. There were 5 Tg + mie per in on ontrol diet, nd Tg + mie per in on NSAIDs (Fig. 8). We found no reltionship etween PGE2 levels nd wter mze performne in Tg + mie on ontrol diet (Fig. 8). However, in Tg + mie given NSAIDs, Frontl Cortex (m Ci/g) we disovered n inverse reltionship etween performne in the wter mze nd PGE2 levels (Fig. 8). Animls performing well in the wter mze hd signifintly lower PGE2 levels thn nimls performing poorly. In ontrst, there were no signifint differenes etween high nd low performers in the levels of TNF-, IL-1 or totl A4 or A42 (Fig. 8 f). Sine elevted PGE2 levels orresponded to poorer memory funtion, we sought to determine whether the pplition of PGE2 to hippompl slies would reverse the enefiil effets of COX-2 inhiitors on A-medited LTP defiits. We found tht 5 mm PGE2 hd no effet on LTP per se, LTP mesuring 154 6% (n = 6), ut loked the ility of the seletive COX-2 inhiitor NS398 to reverse A-medited LTP defiits, LTP mesuring 16 6% (n=6, P5.1) (Fig. 9). The dt indite tht the effet of COX-2 inhiitors on preventing A-medited LTP defiits ours through susequent derese in PGE2 prodution, nd n e reversed y the ddition of exogenous PGE2. Tken together, the results suggest tht the disruption of memory funtion y A is dependent upon COX- 2-medited PGE2 signlling t synpses, whih is loked y NSAIDs. Disussion Our results indite tht loking COX-2 prevents the inhiition of LTP y syntheti, solule A42 nd therefore restores synpti funtion. The dministrtion of NSAIDs tht inhiit COX-2 lone, or COX-1 nd COX-2 together, improves memory funtion in Tg2576 mie, inditing tht memory dysfuntion is lso dependent upon COX-2. There ws no dvntge of iuprofen reltive to nproxen nd MF-triyli. The reovery of memory funtion ws inversely relted to PGE2 levels, ut did not depend upon TNF-, IL-1 or hnges in A. Thus, NSAIDs improved A-indued defiits in memory nd long-lsting plstiity independently of reduing inflmmtion or lowering A42, nd the inhiition of COX-2 ws suffiient to improve synpti plstiity nd memory funtion. Tht NSAIDs influene ytokines other thn TNF- nd IL-1 is possile, ut ws not studied. The involvement of other ytokines or inflmmtory proesses is unlikely, however, euse we demonstrted enefiil effets of NSAIDs t 8.5 to 9 months, n ge prior to the pperne of signifint plques or inflmmtory pthology in Tg2576 mie. We onlude tht neurotoxi inflmmtion leding to miroglil tivtion, TNF- prodution nd IL-1 tivtion plys little, if ny, role in disrupting memory in Tg2576 mie t ges when the plque lod is still modest. Our results do not exlude the possiility tht inflmmtion plys role when the plque urden is greter. Chnges in dendriti spines re elieved to underlie synpti plstiity nd memory. COX-2 is signlling moleule onentrted in dendriti spines (Ymgt et l., 1993; Kufmnn et l., 1996), nd solule A oligomers Downloded from t Trinity College Lirry, Dulin on June 18, 21

11 NSAIDs improve A-medited memory loss Brin (28), 131,651^ () MPS () TNF (pg/mg) (e) Totl A4 (pmol/g) Wter mze proes Tg positive mie inned evenly into thirds: MPS = low, medium, high NSAIDs ind speifilly t dendriti spines (Lor et l., 24). It is intriguing tht COX-2 nd the presumed site of tion of solule A oligomers onverge on dendriti spines. This onvergene ples ntomil onstrints upon possile mehnisms of tion of COX-2 inhiitors on A-medited impirment of memory nd synpti plstiity. Tking into onsidertion this ntomil onstrint, the rpidity of the effets of COX-2 inhiitors nd their reversl y the ddition of exogenous PGE2 on A-medited suppression of LTP in vitro, nd the signifint orreltion etween etter memory funtion nd lower PGE2 levels in vivo, led us to hypothesize novel mehnism y whih NSAIDs my protet ginst Alzheimer s disese. We propose tht NSAIDs lok COX-2-medited prodution of PGE2 within dendriti spines, nd therey prevent the detrimentl effets of A on synpti plstiity nd memory funtion. Our work omplements nd supports reent findings tht COX- 2 potentites the deleterious effets of A on lerning nd memory in trnsgeni mie without hnges in the overll levels of A (Melnikov et l., 26). The modultion of A-indued ehviourl normlities independently of * * NSAIDs NSAIDs PGE2 (pg/mg) IL-1 (fg/mg) Totl A42 (pmol/g) () (d) (f) low MPS medium MPS high MPS NSAIDs hnges in the levels of A is not unpreedented; lueerries ontin sustnes, possily nti-oxidnts, tht improve ehviourl performne in the sene of hnges in myloid lod ut with ttendnt hnges in severl signlling moleules (Joseph et l., 23). The COX-2 inhiitor dose we dministered to mie (2 mg/kg/dy) ws lower thn doses disrupting memory in rts (5 1 mg/kg/olus) (Tether et l., 22). The low doses of the COX-2 inhiitors ppered to lok PGE2 prodution required for solule A oligomers to disrupt memory, ut lrgely spred COX-2 tivity involved in filitting memory, sine NSAID-treted non-trnsgeni mie showed only minor trend towrds impirment. These oservtions suggest tht syntheti, solule A42- nd A 56-medited inhiition of plstiity nd memory re dependent upon sl COX-2, nd tht the enefiil effet of NSAIDs is not direted t suppressing the tivity of indued COX-2, in ontrst to other disese prdigms in whih COX-2 mrna is upregulted in response to injury (Ideol et l., 21). The explntion we fvour for how this might our is tht COX-2, whih is normlly * NSAIDs NSAIDs Fig. 8 Reltionship etween PGE-2, TNF-, IL-1, A levels nd sptil referene memory intg2576 mie. () 13-month trnsgene positive Tg2576 mie (Tg + ) on NSAID or ontrol diets were divided into three evenly sized ins sed upon performne in the wter mze (low, medium nd high MPS tegories). There ws signifint effet of MPS tegory in oth ontrol nd NSAID-treted mie (P5.1 nd P5.1, respetively. :, :, : within ontrol nd NSAID tretment groups represent signifint Fisher s PLSD post ho omprisons. P5.1 y unpired t-test.) () There ws n inverse reltionship etween wter mze performne (MPS) nd ortil PGE-2 levels in NSAID-treted mie (P5.5). P5.1 y Fisher s PLSD. No reltionship etween MPS nd PGE-2 levels ws oserved in mie reeiving diet. (^f) No reltionship ws oserved etween MPS nd hippompl TNF-, hippompl IL-1, ortil totl A4 or ortil totl A42 respetively in ontrol or NSAID-treted mie. Dt re mens SEM. Numers of mie denoted inside rs. Downloded from t Trinity College Lirry, Dulin on June 18, 21

12 662 Brin (28), 131,651^664 L.A.Kotilineket l. Fig. 9 Exogenous PGE2 loks the ility of seletive COX-2 inhiitor to prevent the inhiition of LTP indution y syntheti, solule A42. () LTP indued y single rief high-frequeny stimultion (HFS) (losed irles) nd LTP indution in the presene of PGE2 (5 mm), pplied 4 min prior to HFS (open irles). PGE2 t this onentrtion hs no effet on LTP. () The COX-2 inhiitor NS398(2 mm), pplied 6 min prior to HFS prevents the A-medited inhiition of LTP indution (losed irles), ut is unle to do so in the presene of 5 mmpge2 (open irles). present in dendriti spines, plys permissive role in the inhiition of plstiity nd memory y syntheti, solule A42 nd A 56. Another possile explntion would involve hnge in the synpti omprtmentliztion of COX-2 in response to solule A, without n overll inrese in COX-2 levels, or the existene of distint pools of COX-2 in seprte mirodomins of dendriti spines one pool tht filittes LTP nd memory in response to glutmte inding t NMDA reeptors, nd nother pool tht disrupts LTP nd memory in response to A inding to dendriti spines. A third explntion would e the ourrene of n inrese in COX-2 or PGE2 turnover in synpses or dendrites, without drmti hnge in the levels of COX-2 or PGE2. It hs een suggested tht NSAIDs protet ginst Alzheimer s disese y reduing pro-inflmmtory ytokine prodution y inhiiting COX or tivting PPAR-g (Lehmnn et l., 1997; Coms et l., 2). It hs lso een proposed tht modultion of g-seretse my e importnt (Eriksen et l., 23; Weggen et l., 21). We found no evidene for the involvement of these mehnisms in improving A-indued defiits in memory or synpti plstiity, whih hs never een speifilly exmined efore. Importntly, the NSAIDs tht were not seletive A42-lowering gents (SALAs) were s effetive t resuing memory nd LTP s iuprofen, SALA (lthough A42 levels were unffeted y iuprofen under the onditions of our study). Our results re supported y reent nlysis of pooled dt from six prospetive epidemiologil studies, whih provided the sttistil power required to ddress speifilly the impt of SALAs on the prevention of Alzheimer s disese, showing no dvntge of SALAs in onferring protetion ginst Alzheimer s disese (Szekely et l., 28). Why hve NSAIDs, speifilly oxis, een disppointing in the tretment of ptients? In Alzheimer s disese, COX-2 levels progressively inrese (Ho et l., 21), proly sustined y positive feedk loops involving A, glutmte nd inflmmtory ytokines. Elderly people on high-dose NSAIDs re more likely to hve memory deteriortion, possily inditive of their inresed suseptiility to the toxi effets of inhiiting physiologil COX-2 (Hnlon et l., 1997). Chroni COX-2 elevtion might lso led to ellulr hnges tht re unresponsive to NSAIDs (Dore et l., 23). Furthermore, COX-2 inhiition my fil to lok other pthologil sdes downstrem of A, like those involving tu, whih ould ply signifint roles in the pthogenesis of lter stges of Alzheimer s disese (Lewis et l., 21; Oddo et l., 24; SntCruz et l., 25; Roerson et l., 27). This would explin why NSAIDs hve lrgely filed to slow Alzheimer s disese progression in linil trils (reviewed in MGeer nd MGeer, 27), why NSAIDs fil to prevent Alzheimer s disese in elderly individuls 47 yers of ge (Group et l., 27), nd lso why NSAIDs pper to exert more roust protetion ginst Alzheimer s disese in individuls who were exposed severl yers prior to the onset of symptoms in epidemiologil studies (Stewrt et l., 1997; in t Veld et l., 21; Zndi et l., 22). Our results predit tht the predominnt effiy of NSAIDs will e found in individuls with norml levels of COX-2, prior to inreses in COX-2 due to inflmmtion or glutmte. Brodening investigtions on the effets of NSAIDs to involve synpse nd rin funtion my renew interest in other potentil protetive mehnisms of NSAIDs, thus widening the possiilities for developing sfe prophylti meditions for Alzheimer s disese. Downloded from t Trinity College Lirry, Dulin on June 18, 21

13 NSAIDs improve A-medited memory loss Brin (28), 131,651^ Supplementry mteril Supplementry mteril is ville t Brin online. Aknowledgements We grtefully knowledge Deirdre Cooper-Blketer, Jen Pulson, Din Nsh, Jennifer Lng, Aron Guimres, Stephen Csper, Brin Clrk nd Mthew Shermn for help with niml reeding, genotyping nd ehviourl testing, Dniel Fdle for tehnil ssistne, Mike Kuskowski for help with sttistil nlysis nd Kthleen Zhs, John Breitner, Pul Chpmn nd Riley MCrten for ritil disussions. We thnk Merk & Co. In. for providing M-F triyli how nd Peppi Prsit nd Weiho Chen t Merk & Co. In. for mesuring levels of rin nd serum iuprofen, nproxen nd M-F triyli. We delre no ompeting interests. Supported y NIH grnts R1- NS33249 (K.H.A.), R1-MH65465 (K.H.A.), P1-AG15453 (P.F.C., S.G.Y. nd K.H.A.), nd PO1-AG18357 (G.Y.S. nd A.S.), the Tulloh Endowment (K.H.A.), R1-AG13471 (G.M.C.) nd the Wellome Trust (R.A.). Funding to py the Open Aess pulition hrges for this rtile ws provided y the Minnesot Aging nd Alzheimer s Reserh Endowment. Referenes Ashe KH. Mehnisms of memory loss in Aet nd tu mouse models. Biohem So Trns 25; 33: Bik EJ, Kim EJ, Lee SH, Moon C. Cylooxygense-2 seletive inhiitors ggrvte kini id indued seizure nd neuronl ell deth in the hippompus. Brin Res 1999; 843: Bzn NG. COX-2 s multifuntionl neuronl modultor. Nt Med 21; 7: Broe GA, Gryson DA, Cresey HM, Wite LM, Csey BJ, Bennett HP, et l. Anti-inflmmtory drugs protet ginst Alzheimer disese t low doses. Arh Neurol 2; 57: Celos R, Alvrez XA, Fernndez-Novo L, Frno A, Mngues R, Pellier A, et l. Brin interleukin-1 et in Alzheimer s disese nd vsulr dementi. Methods Find Exp Clin Phrmol 1994; 16: Chpmn PF, White GL, Jones MW, Cooper-Blketer D, Mrshll VJ, Irizrry M, et l. Impired synpti plstiity nd lerning in ged myloid preursor protein trnsgeni mie. Nt Neurosi 1999; 2: Chen G, Chen KS, Knox J, Inglis J, Bernrd A, Mrtin SJ, et l. 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