Research Article Role of Copper and Cholesterol Association in the Neurodegenerative Process

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1 Interntionl Journl of Alzheimer s Disese Volume 2013, Artile ID , 15 pges Reserh Artile Role of Copper nd Cholesterol Assoition in the Neurodegenertive Proess Nthlie Arnl, 1 Gustvo R. Morel, 1 Mrí J. T. de Alniz, 1 Omr Cstillo, 2 nd Crlos A. Mrr 1 1 INIBIOLP (Instituto de Investigiones Bioquímis de L Plt), CCT L Plt, CONICET-UNLP, Cátedr de Bioquími y Biologí Moleulr, Fultd de Cienis Médis, Universidd Nionl de L Plt, 60 y 120 (1900) L Plt, Argentin 2 CIC (Centro de Investigiones Crdiovsulres), CCT L Plt, CONICET-UNLP, Fultd de Cienis Médis, UniversiddNionldeLPlt,60y120(1900)LPlt,Argentin Correspondene should e ddressed to Crlos A. Mrr; [email protected] Reeived 7 July 2013; Revised 5 Septemer 2013; Aepted 5 Septemer 2013 Ademi Editor: Rosnn Squitti Copyright 2013 Nthlie Arnl et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Age is one ofthe min ftors involved inthe development of neurologil illnesses, in prtiulr, Alzheimer, nd it is widely held tht the rpid ging of the world popultion is ompnied y rise in the prevlene nd inidene of Alzheimer disese. However, evidene from reent dedes indites tht Cu nd Cho overlod re emerging ustive ftors in neurodegenertion, hypothesis tht hs een prtilly investigted in experimentl models. The link etween these two vriles nd the onset of Alzheimer disese hs opened up interesting new possiilities requiring more in-depth nlysis. The im of the present study ws therefore to investigte the effet of the ssoition of Cu + Cho (CuCho) s possile synergisti ftor in the development of n Alzheimer-like pthology in Wistr rts. We mesured totl- nd noneruloplsmin-ound Cu nd Cho (free nd sterified) ontents in plsm nd rin zones (ortex nd hippompus), mrkers of oxidtive stress dmge, inflmmtion, nd progrmmed ell deth (spse-3 nd lpin isoforms). The rtio et-myloid (1-42)/(1-40) ws determined in plsm nd rin s neurodegenertive iomrker. An evlution of visuosptil memory (Brnes mze test) ws lso performed. The results demonstrte the estlishment of prooxidtive nd proinflmmtory environment fter CuCho tretment, hllmrked y inresed TBARS, protein ronyls, nd nitrite plus nitrte levels in plsm nd rin zones (ortex nd hippompus) with onsequent inrese in the tivity of lpins nd no signifint hnges in spse-3. A simultneous inrese in the plsm Aβ1-42/Aβ1-40 rtio ws found. Furthermore, slight ut notiele hnge in visuosptil memory ws oserved in rts treted with CuCho. We onlude tht our model ould reflet n initil stge of neurodegenertion in whih Cu nd Cho intert with one nother to exerte neurologil dmge. 1. Introdution The ging of the world popultion is eing ompnied y rise in the prevlene nd inidene of Alzheimer disese (AD) nd other neurodegenertive illnesses. In the USA, the numer of ptients with AD is expeted to inrese to 13 million y 2050 nd in the Europen Union (EU) to over 4 million [1]. The mortlity rte of AD is seond only to ner nd stroke. Epidemiologi dt indites tht the world popultion will hve grown onsiderly y 2025 nd the perentge of elderly people will e signifintly higher [2]. Sine ge is one of the mjor risk ftors in the development of AD, it is expeted tht the inidene of this disese will lso inrese. However, experimentl evidene detiled in review y Brewer indites tht other ftors in ddition to ge ould ply ritil role[3] in the development of diseses suh s Alzheimer nd tht the ontriution of inorgni opper (Cu) hs een underestimted. It is widely known tht more thn 95% of AD ses re spordi nd only 2 7% re genetilly determined [4]. Thus, ny environmentl ftorslikelytohvenetiopthogeniroleinadshoulde investigted. Though Cu is essentil to humn helth, Cu overlod hs een ssoited with mentl deline [5] ndprtiulrly with AD development [3, 6, 7]. Dt from Squitti s group speifilly demonstrted tht free Cu (lso known s NCBC

2 2 Interntionl Journl of Alzheimer s Disese or noneruloplsmin-ound Cu) is elevted in the lood of AD ptients, negtively orreltes with ognition, nd predits the rte of loss of ognition [8 10]. More reently, our group orroorted these findings in n independent humn ohort nd demonstrted tht inresed NCBC hs diret impt on the disese durtion [11]. We lso proposed the NCBC/eruloplsmin rtio s preditive mrker of risk for the first-degree reltives of AD ptients. Sprks nd Shreurs [12] first demonstrted tht Cu supplementtion in drinking wter given to rits under diet with exess holesterol (Cho) produed n indution of β-myloid plques nd lerning defiit. In ddition, Lu et l. reported tht tre mounts of Cu tivte the poptoti sde nd exerte et myloid-indued neurotoxiity in Cho-fed mie through TNF-medited inflmmtory pthwy [13, 14]. Very reently, Brewer hs reviewed the theory of inorgni Cu toxiity in Alzheimer disese s ustive ftor in ognitive loss [6]. However, the question of how inorgni Cu might trigger neurodegenertive proess is still mtter of dete [15]. The degree of exposure of humn popultions to Cu is lso ontroversil issue. There is little dt on Cu overlod in humns sine most of the ville evidene (experimentl or epidemiologil) ws otined from niml models. However, the regultory frmework for hroni Cu exposure in lrge humn popultions indites tht pollution, drinking wter, nd dietry Cu-ontining supplements re the min soures of exposure [6, 16]. The dietry referene intke for people in the USA, United Kingdom, Europe, nd Austrli vries from 0.16 to 0.98 (Estimted Averge Requirements) EAR or (Reommended Dietry Allowne) RDA expressed in mg Cu/kg ody weight, with onsiderle vritions s funtion of ge. The (Popultion Referene Intke) PRI ws reported etween 0.3 nd 1.5 mg Cu/kg ody weight [16]; however, these limits were lrgely surpssed in mny irumstnes suh s ingestion of fish, ivlves, or ontminted drinking wter [3, 17]. In umulted dt on 280 smples ofhouseholddrinkingwterllrossnorthameri,72% of the smples hve Cu levels ove those enhning AD in experimentl models [3]. The ontent of inorgni Cu in dietry supplements n e s high s 3 mg/pill (pprox. 2 mg ove the EAR or RDA). Furthermore, we nd others hve demonstrted signifintly higher Cu levels in plsm of women using Cu-IUDs nd in lood smples from frmers working with Cu-sed pestiides [17 19]. Most importntly, we found ltertions in Cu homeostti iomrkers in neurodegenertive ptients nd their first-degrees reltives [11]. Ft ingestion hs lso een linked to AD prevlene [20]. Speifilly, Cho hs een ssoited with oxidtive stress nd AD development [21, 22], with most of the experimentl evidene emerging from explortion of the role of Cho in βmyloid formtion [23]. Despite the puity of epidemiologil dt from humn studies, it is resonle to ssume tht Cho plys t lest some role in lerning nd memory nd is ssoited with AD pthogenesis [24]. Of prtiulr interest is evidene showing tht Cu gretly exertes ognitive deline in those people inluded in the highest quintile of ft ingestion [25]. Experimentl evidene otined from rit nd mouse models suggests tht the ssoition of CundChoneriskftorforADdevelopment[12 14]. Moreover, it ws hypothesized tht Cu ould oxidize Cho, generting sustnes toxi to the rin [3]. However, the mehnism of tion of Cu nd Cho in AD inidene nd development is poorly understood nd requires further investigtion. Thus, the im of this work ws to study the effets of Cu nd Cho ssoition on the two min rin res ffeted in AD, ortex nd hippompus, using model of Wistr rts. Speifilly, we imed to determine for eh nutritionl supplement lone or in omintion (i) the pity to instll oxidtive/nitrtive dmge; (ii) hnges in the levels of the min ntioxidnt moleules (glutthione nd α-toopherol); (iii) the possile development of proinflmmtory ondition y nlyzing the onentrtion of prostglndins PGE2 nd PGF2α; (iv) the tivities of the two min protese systems ssoited with progrmmed ell deth, spse-3 nd lpins (μ- nd m-); nd (v) possile hngesinvisuosptilmemorysssessedymensofthe Brnes mze test. Our findings ould e useful in further investigting the mehnisms underlying the neurodegenertive proess nd lso in lolizing puttive trgets for preventive interventions ssoited with endogenous nd/or exogenous ustive ftors suh s Cu nd Cho, lone or in omintion. 2. Mteril nd Methods 2.1. Chemils. All hemils used were of nlytil grde nd otined from Sigm Chem. Co. (Buenos Aires, Argentin or USA), Merk (Drmstdt, Germny), nd Crlo Er (Miln, Itly) Animls nd Tretments. Certified pthogen-free mle Wistr rts were used. The rts were mintined t ontrolled temperture (25 C) nd reltive humidity of 60% with fored ventiltion, under norml photoperiod of 12 h drkness nd 12 h light. The helth of the nimls ws monitored in ordne with the interntionlly reommended prties of the (Institute of Lortory Animl Resoures, Commission of Life Sienes, Ntionl Reserh Counil) ILAR. Solid food nd drinking wter were provided d liitum. The diets for the experiments were prepred in our lortory ording to the reommendtions for Wistr rts [26]. All proedures for hndling the nimls followed the NIH regultions [27]. The experimentl protool ws reviewed nd pproved y (Bioethis Committee of the Fulty of Medil Sienes, UNLP) COBIMED under the ode#00382/ Experimentl Protools. Rts (21 dys old) were rndomly ssigned (ten nimls per group) to the protools detiled s follows nd treted during eight weeks. The ontrol group (C) ws mintined on l-prepred pellets s reommended for norml growth, ontining 7 ppm of Cu [28, 29]. The Cusupplemented experimentl group (Cu) ws fed on ontrol pellets nd tp wter supplemented with 3 mg/l (or ppm) of Cu in the form of ultrpure CuSO 4 (Merk, Drmstdt, Germny),theCho-supplementedgroup(Cho)wsfedonpellets ontining 2% (W/W) of Cho (87% pure) (otined from

3 Interntionl Journl of Alzheimer s Disese 3 Sporiti SRL, Buenos Aires, Argentin), nd the Cu + Chosupplemented group (CuCho) ws simultneously treted with Cu in wter + Cho in food. Rts were monitored during the experimentl period to oserve their ehvior, quntify wter, nd food onsumption nd determine their ody weight gin. Totl Cu onentrtion in tp wter supplemented with CuSO 4 ws determined y mens of tomi sorption methodology nd ws 3.42 ± 0.21 ppm (mens of ll dily mesurements long the experimentl period). Considering tht eh niml imied etween 4.9 ± 0.4 nd 15.0 ± 1.1 mlwter/dy(ttheeginningndtheendofthe protool, resp.), mximum of 0.01 to 0.05 mg Cu/dy ws quired from wter ( dose equivlent to 0.06 nd 0.18 mg Cu/Kg live niml, resp.). Liner regression urves nd ANOVA test for Cu ontent in food demonstrted tht there were no signifint vritions etween the 6 preprtions used for the experiments (7.22 ± 0.31 ppm or mg Cu/Kg diet). Fe nd Zn ontent (determined y tomi sorption spetrometry) were the sme in ll preprtions (45.9 ± 1.8 nd 66.6 ± 2.0 ppm, resp.). Ingestion of solid food long the experiments vried from 11.6±0.8 to 29.7±2.8 g/rt, implying tht the orl ingestion of Cu ws in the rnge of 0.08 to 0.21 mg Cu/dy/rt (0.90 to 1.21 mg Cu/Kg live niml, men of 1.06 ± 0.11 mg Cu/Kg) Smple Colletion. At the end of the tretments, nimls were deeply nesthetized with ketmine (70 mg/kg) nd xylzine (5 mg/kg) pplied intrmusulrly nd then srified y depittion. Brins were rpidly tken out nd disseted in two zones, ortex nd hippompus, using the tls of Pxinos nd Wtson [30] s guide for tissue dissetion nd Binoulr Stereosopi Arno Ztx-1065 Mirosope (Instrumentl Psteur, Buenos Aires, Argentin). Both rin regions were wshed, weighed, nd homogenized using uffer Tris/HCl (10 mm ph 7.4) with surose (70 mm), mnnitol (230 mm), ethylenediminetetreti id (EDTA) (1 mm), nd dithiothreitol (DTT) (1 mm) Atomi Asorption Mesurements. Aliquots of smple were digested with mixture of 4 ml of HNO 3 () nd 1mL HClO 4 (Aldrih or Sigm Chem. Co., Buenos Aires, Argentin) y heting t 120 C for 80 min in minerliztion lok [31]. The digests were ooled, diluted with ultrpure wter (18 mω m, Crlo Er, Miln, Itly), nd ultrfiltered y 0.22 μm Millipore memrne (Milli-Q Purifition System, from Millipore, CA, USA). Ultrfiltered dissolutions were diretly spirted into the flme of Perkin-Elmer 1100 B Spetrophotometer equipped with Perkin-Elmer thode lmp (Perkin-Elmer Corp., Norwlk, CT, USA) t spetrl width of 1 nm. Stndrd solutions of 100 ppm from HCR In. (QuimiNet, Buenos Aires, Argentin) were used for Zn nd Fe. Cu determintions were lirted with stndrd solution (200 ppm) of Cu (NO 3 ) 2 in HNO N (Tritrisol from Merk Co., Drmstdt, Germny). All mesurementswererriedoutinpekheightmode(324.7nm line). The intr-[(sd/x) 100] nd inter-[(δsd/δx) 100] ssy oeffiients of vrition were 15.5 nd 6.0%, respetively. We routinely otined similr eqution for the lirtion urve (IR = [Cu, mg/l]), nd the sttistil nlyses demonstrted orreltion oeffiient lwys etween 0.95 nd In ddition, we explored the so-lled mtrix effets tht ould hve modified the slopes of the stndrd regressions. In spiked smples the otined vlues vrying from 48 to were very similr to those of Cu stndrd solutions, inditing tht the mtrix effet ws not signifint or ws negligile. The men for reovery nd RSD for spiked smples ws 99.7% nd 3.3%, respetively, nd the detetion limit ws 0.09 mg/l. In order to verify the ury of the method, we explored the influene of time fter dilution, temperture of id digestion, nd onentrtion of HNO 3 /HClO 4 following the suggestions of Terrés-Mrtos et l. [32]. We lso heked our results with iologil smples (plsm nd homogentes) ginst Seronorm Tre Elements Serum (from Sero Ls, Billingstd, Norwy) nd found no signifint differenes etween the otined nd the delred (ertified) onentrtions Ceruloplsmin (CRP) Levels nd Noneruloplsmin- Bound Copper (NCBC). Smples were nlyzed y the enzyme onversion of p-phenylenedimine into lue-olored produt [33] whih ws then mesured t 550 nm. Retion proeeded t 37 C in uffer glil eti/sodium ette (50 mm, ph 5.5) diretly into flt-ottomed pltes, using Miroplte Reder SpetrMx M2/m2 e model from Moleulr Devies Anlytil Tehnologies (Sunnyvle, CA, USA) for 3 min. Intr- nd interssy oeffiients of vrition were 8.3 nd 4.4%, respetively. CRP onentrtions were lulted y omprison with the retion rte of pure CRP stndrd (Sigm Chem. Co., Buenos Aires, Argentin). Using the Cu nd CRP dt, we lulted the non-crp-ound Cu (NCBC, or so-lled free Cu) s desried y Brewer [3] y sutrting the mount of Cu ound to eh mg of CRP from dt of totl Cu. This prmeter n e esily expressed in perentges using the formul (([Cu]-47.2 [CRP]) 100/[Cu]), where Cu is in μmol/l nd CRP is in g/l [34] Lipid Anlysis. Totl lipids were extrted y the method of Folh et l. [35]. Aliquots of this solution were tken to mesure totl Cho y n enzymti method using ommeril kit from Wienner L (Rosrio, Argentin). To estimte the mount of esterified Cho (ECho), liquots of the lipid extrts were seeded in high-resolution pre-oted sili gel pltes (10 20 m) with onentrtion zone for thin lyer hromtogrphy (Whtmn Adsorption 60 Å Sili Gel HP-TLC Pltes, CA, USA) nd developed with diethyl ether : hexne : eti id (90 : 4 : 1, y vol) s desried elsewhere [36]. AuthentistndrdsofChondCho-esthers (from Avnti Polr Lipids, Ontrio, Cnd) were run in prllelndreveledyiodinevpors.identifiedzoneswere srped off the pltes, eluted with Folh retive, evported, dissolved in 50mM phosphte uffer (ph 7.4) with 1% sodium deoxyholte, nd enzymtilly nlyzed using the ommeril kits from Wienner L (Rosrio, Argentin) Biomrkers of ROS Prodution. Thiorituri idretive sustnes (TBARS) were mesured in rin homogentes s previously desried[37]. TBARS (minly mlondildehyde, MDA) reted with 2-thiorituri id (TBA)

4 4 Interntionl Journl of Alzheimer s Disese to yield TBA-MDA dduts whih were quntified t 532 nm. The onentrtion of the hromophore ws lulted from lirtion urve prepred with fresh tetrmethoxypropne (TMP) solutions (TMP ws purhsed from Sigm Chem. Co., Buenos Aires, Argentin). Nitrte nd nitrite [NOx] onentrtion were mesured using the method of Griess on smples previously redued with vndium hlorohydrte ording to Mirnd et l. [38]. Quntifition ws performed fter lirtion with stndrd solutions of sodium nitrte from Merk Co. (Drmstdt, Germny). Protein ronyls (PCs) were determined y the method of Reznik nd Pker [39]. The onentrtions of PCs were lulted from lirtion urve prepred with stok solution of sodium pyruvte (Sigm Chem. Co.) Antioxidnt Defense System Redued (GSH) nd Oxidized (GSSG) Glutthione Content. Totl glutthione ws determined y the glutthione redutse/dithionitroenzoi (DTNB) method tht n mesure oth GSH nd GSSG [40]. To lulte the GSH/GSSG rtio, smples were renlyzed fter derivtiztion with divinylpyridine (3 mm finl onentrtion) Vitmin E (α-toopherol). Vitmin E (α-toopherol) wsmesuredfterextrtionwiththebuttrissnddiplok method [41] using the HPLC tehnique of Bgnti et l. [42] in reverse phse/c-18 sili olumn from ALLTECH Assoites,In.(Deerfield,IL,USA).TheECONOSILC 18 olumn with Diret-onnet Crtridge Gurd Column System ws operted t mximum pressure of 500 psi in HithiHPLCSystem(Tokyo,Jpn).Themountofvitmin ws eletronilly lulted using internl lirtion with pure α-toopherol (Sigm, Bs. As.), nd the results were expressed in μm onentrtion of α-toopherol Glutthione Redutse (GR). GR tivity ws determined y the method of Crlerg nd Mnnervik [43]. The speifi tivity of the enzyme ws lulted for eh smple in terms of U/min mg protein (ε = 6.22 nm 1 m 1 for sorne t 340 nm) Biomrkers of Inflmmtion. Prostglndin F2α (PG F2α) nd prostglndin E2 (PGE2) were mesured using the PGF2α EIA Kit nd PGE2 Express EIA Kit, respetively (Cymn, Migliore Llustr S.R). The results were expressed s ng of eh prostglndin/mg totl protein Progrmmed Cell Deth Biomrkers Cspse-3 Ativity. Cspse-3 tivity ws mesured y olorimetri ssy kit (CASP-3-C) sed on the hydrolysis of the syntheti peptide sustrte etyl-asp-glu-vl-asp-pnitroniline (A-DEVD-pNA) y spse-3 (Sigm Chem. Co., Buenos Aires, Argentin). The resulting p-nitroniline (p-na) ws monitored t 405 nm. Eh ssy ws run in prllel with inhiitor-treted homogentes (to mesure the nonspeifi hydrolysis of the sustrte) nd spse-3 positive ontrol (using ommeril spse-3, 5 mg/ml provided y the kit mnufturer). A lirtion urve using stndrd solution of p-nitroniline (p-na) ws lso run for eh ssy to lulte the tivity of the protese expressed s μmol p- NA relesed/min mg protein Milli- (m-) nd Miro- (μ-) Clpins. The ssy involves the hydrolysis of whole ultrpure sein (Sigm, Chem. Co., CA, USA) y lpin tivity nd the susequent detetion of trihloroeti id (TCA) solule peptidi frgments t 280 nm [44]. To selet the tivity of the lpin isoforms, the level of lium in the medium ws regulted (5 mm or 500 μm of CCl 2 for m- or μ-lpin, resp.). The tivity of lpins ws lulted onsidering tht unit of lpin is the mount of enzyme tht produes hnge of sorne of 0.01 t 280 nm. Results were expressed s units/min mg of protein Mrkers of Neurodegenertion Bet Amyloid Peptides (1-40) nd (1-42). Bet myloid peptides (Aβ) (1-40) nd (1-42) were mesured using Humn/Rt β Amyloid-40 ELISA kit Wko II nd the Humn Amyloid-42 ELISA kit Wko High-Sensitivity, respetively. The Aβ(1-42)/(1-40) rtio ws then lulted from the individul dt expressed s piomole of the respetive Aβ peptide/mg totl protein Brnes Mze Test. The Brnes mze is lk ryli irulr pltform, 122 m in dimeter nd elevted 108 m off the floor, ontining twenty holes round the periphery. The 10 m dimeter holes re uniform in pperne ut, one hole is onneted to n espe ox, onsisting of 38.7 m long 12.1 m wide 14.2 depth m removle ox. Four proximl visul ues (30 m high, opque lk geometri figures: ross, irle, squre, nd tringle) were loted intheroom50mfromtheirulrpltform.theespe ox remined in fixed position reltive to the ues, to ensure rndomiztion of the hole ssoited with the espe tunnel. In the enter of the pltform is strting hmer (n opque,26mdimeter,20mhigh,ndwhiteplstiopenended ylinder). A 90 db white-noise genertor nd whitelight 500 W ul provided motivtion for esping from the pltform. The quisition session nd the proe tril session were performed on the sme dy. In rief, the experiment onsisted of eight quisition trils (t1 t8) followed y single evlution tril (t9). Aquisition trils egn with the niml inside the strting hmer for 30 seonds in the presene of onstnt uzz. The hmer ws then rised, the versive stimulus (intense right light) ws swithed on, nd the rt ws llowed to freely explore the mze. The rts were eh given120stolotetheorrethole.ifytheendofthis periodtheyhdnotenteredtheespeoxoftheirown ord,theyweregentlypikedupndpledoverthehole ove the espe ox. The evlution tril proeeded in the sme mnner s desried ove ut without the strt ox. At the end of eh tril, the versive stimulus ws swithed off, the rt remined on the espe ox during 60 s, nd the white light ws swithed off. In order to eliminte ny possile olftory lues from the mze, it ws lened with

5 Interntionl Journl of Alzheimer s Disese 5 Tle 1: Copper (Cu) levels in nimls fed with the experimentl diets. Cu (ng/mg prot) Plsm Brin Diets Cortex Hippompus Totl NCBC Totl NCBC Totl NCBC Control 18.5 ± ± ± ± ± ± 0.04 Cu 23.2 ± ± ± ± ± ± 0.02 Cho 18.1 ± ± ± ± ± ± 0.04 CuCho 22.9 ± ± ± ± ± ± 0.03 Cu ontent ws determined fter minerl digestion of the smples y tomi sorption spetrometry s desried in Setion 2.5. Results re the men of 10 independent mesurements nlyzed in triplite ± SD.Comprisons etween dtwereperformed yanova+tukey testtp < Sttistil differenes mong the experimentl diets were indited with distint supersript letters (vlues within the sme olumnwith different supersript lettersresttistilly signifint etween them). Tle 2: Esterfied (ECho) nd free (FCho) holesterol levels in nimls fed with the experimentl diets. Cho (nmoles/mg prot) Diets Plsm Brin zones Cortex Hippompus Esterified Free FCho/Eho Esterified Free FCho/Eho Esterified Free FCho/Eho C 16.4 ± ± ± ± ± ± ± ± ± 0.60 Cu 16.8 ± ± ± ± ± ± ± ± ± 0.33 Cho 22.2 ± ± ± ± ± ± ± ± ± 0.21 CuCho 18.3 ± ± ± 0.05 d 70.0 ± ± ± ± ± ± 0.11 d Cho nd Cho-esters ontents were determined enzymtilly fter HP-TLC s desried in Setion 2.7. Results re the men of 10 independent mesurements nlyzed in triplite ± SD. Comprisons etween dt were performed y ANOVA + Tukey test t P < Sttistil differenes mong the experimentl diets were indited with distint supersript letters (vlues within the sme olumn with different supersript letters re sttistilly signifint etween them). 10% ethyl lohol solution t the eginning of the 15 min intertril period. An individul hole explortion ws defined s eing single downwrd hed defletion towrd the inside of the hole. The following prmeters were determined: (i) first-hole lteny time (in s) spent y the niml etween eing relesed from the strt ox nd exploring hole in the mze for the first time; (ii) espe-ox lteny time (defined in the quisition nd retention test trils s the time (in s) spent y the niml etween eing relesed from the strt ox nd entering the espe ox nd, in the se of the preferene test nd extintion trils, the time elpsed efore the first explortion of the espe hole); nd (iii) nongol hole explortion (defined s the numer of explortions of holes other thn the espe hole, the explortions eing onsidered s errors during the quisition nd proe trils). In the se of the evlution trils, we evluted the hole explortion frequeny (the numer of explortions of eh hole during the tril in whih the espe hole ws numered s hole 0 for normlized grphil representtion purposes, 1 to 10 lokwise, nd 1 to 9 ounterlokwise). The ehviorl mesurements were reorded using video mer mounted 110 m ove the pltform, linked to omputer. The video performnes of eh rt were nlyzed using the video nlysis softwre Kinove-Cretive Commons Attriution (v 0.7.6) Sttistil Anlysis. Allvluesrepresentthemenof 6 rts ssyed in triplite expressed s men ± stndrd devition (SD). Dt were nlyzed y ANOVA plus Tukey test with the id of SPSS softwre (SPSS In., Chigo, IL).TonlyzethedtfromtheBrnesmzetest,multiple omprisons were drwn with the ontrol group using twowy ANOVA plus the Holm-Sidk post ho test t two levels of signifine (P < 0.05 nd 0.01). Dt were lso nlyzed usingmanovawithidentilfinlonlusions.results were lso plotted nd nlyzed using Sigm Sientifi Grphing Softwre (version 11.0) from Sigm Chem. Co. (St. Louis, MO). The sttistil signifine of differenes is indited y distint supersript letters (dt with distint supersript letters re sttistilly different (P < 0.01) etween them). 3. Results 3.1. Cholesterol nd Copper Levels. Tle 1 shows the levels of totl nd free Cu (NCBC or noneruloplsmin-ound Cu)inplsmndinrinortexndhippompusfter the experimentl tretments. Plsm totl Cu nd NCBC exhiited disrete (ut sttistilly signifint) inreses in those groups reeiving Cu (Cu nd CuCho). Totl Cu lso inresedinothortexndhippompusftercusupplementtion (lone or in omintion with Cho). Cortex shows nonsignifint tendeny towrds higher NCBC fter Cu or CuChotretments,whereshippompusshowssignifint inrese in this prmeter with respet to the ontrol group. TheresultsoftheChonlysis(freendesterified)re shown in Tle 2, from whih it is evident tht tissue from

6 6 Interntionl Journl of Alzheimer s Disese nmoles (mg.prot.) Control Cu Cho CuCho TBARS PCs NOx nmoles (mg.prot.) () () Figure 1: Oxidtive/nitrtive stress iomrkers in plsm () nd rin ortex nd hippompus (). Lipid peroxidtion (TBARS) (nmol MDA/mg protein), protein oxidtion (PCs) (nmol MDA/mg protein), nd onentrtion of nitrites nd nitrtes ([NOx]) (μmol/mg protein) were determined in plsm nd rin ortex nd hippompus following the proedures desried in desried in Mteril nd Methods Setion. Tretments re indited with different olors. Control (white rs), Cu (gry rs), Cho (drk gry rs), nd CuCho (lk rs). Results re expressed s men of 10 rts ssyed in triplite ± stndrd devition (SD). Signifint differenes were indited y letters (dt with distint letters re sttistilly different etween them t P < 0.01) C H C H C H TBARS PCs NOx Control Cu Cho CuCho the entrl nervous system hs prtiulr Cho metolism hrterized y higher levels of free Cho (25 times higher thn in the se of plsm). Smples from rin nd plsm ehve differently from one nother. Cu ddition to drinking wter produed no signifint hnges in plsm totl Cho nd slight inrese in ortex nd hippompus t the expense of the esterified form (ECho). Furthermore, CudidnothngethertioetweenfreendEChoin plsm smples ut did lower this prmeter in the rin homogentes. Supplementing food with Cho used higher level of totl Cho in peripherl plsm (from 27.0 ± 0.5 in the ontrol group to 33.7 ± 0.6 nmoles/mg protein in Chosupplemented rts) with slight ut signifint derese in the proportion of free/eho. In rin, ll the tretments led to n inrese in totl (free + esterified) Cho with respet to ontrol dt, though the inrese ws sustntilly higher nd differentilly orhestrted in those groups reeiving Cho supplementtion. In the se of Cho-treted rts, this ourred t the expense of free Cho; however, in rts under CuCho tretment, it ws t the expense of the ECho form. Under CuCho tretment, hippompus rehed even higher vlues of totl Cho thn ortex (pprox. 311 versus 295 nmoles/mg protein). Addition of Cu lone signifintly elevted the ECho in oth rin tissues, more notiely in hippompus thn in ortex. Another differentil result for the CuCho group ws derese in free Cho with respet to ontrol rts in ortex, phenomenon not oserved in hippompus. The resultsotinedforthertiosetweenfreendechostrongly suggest tht Cu ddition ws le to modify the lne of these two prmeters, triggering the umultion of ECho in ortex nd hippompus, perhps filitting its esterifition or impeding its degrdtion, or oth. This effet is espeilly notiele under onditions of simultneous Cu nd Cho overlod nd, even more interestingly, ws ontrry to the effet oserved in peripherl plsm smples Biomrkers of Oxidtive Stress. In order to detet whether inorgni Cu in drinking wter nd Cho in food produed oxidtive/nitrtive stress, we mesured the oxidtion end produts of lipids (TBARS) nd proteins (PCs) nd lso the levels of NOx (nitrtes nd nitrites derived from the spontneous dismuttion of nitri oxide) s iomrkers of dmge. Levels of the three mrkers nlyzed were higher fter CuCho tretment oth in plsm nd in rin ortex nd hippompus (Figures 1() nd 1()). In plsm, tretment with Cu lone inresed TBARS levels (Figure 1()), wheres ll three iomrkers were signifintly inresed fter CuCho osupplementtion. Brin homogentes exhiited similr results, with inresed levels of ll three iomrkers fter Cu or CuCho supplementtion (with the solo exeption of PCs in oth rin regions, whih inresed only with simultneous exposuretocundcho). We lso mesured the levels of the two min ntioxidnt moleules for wter- nd lipid-solule ellulr omprtments (totl glutthione-gsh+gssg- nd α-toopherol, resp.) (Tle 3).Plsmlevelsoftotlglutthionewerehigher in rts fed on Cu plus Cho supplements. In the sme experimentl group (CuCho), the α-toopherol onentrtion ws 27% lower thn in the ontrol dt. In rin ortex, we lso oserved n inrese in totl glutthione fter CuCho tretments nd simultneous inrement in the GSSG/GSH rtio s onsequene of higher level of GSSG. Conomitntly, the tivity of glutthione redutse (GR) in ortex ws

7 Interntionl Journl of Alzheimer s Disese 7 Tle 3: Totl glutthione ontent, rtio oxidized (GSSG)/redued (GSH) glutthione, glutthione redutse tivity (GR), nd onentrtion of α-toopherol (α-to) in plsm nd rin ortex nd hippompus from the different experimentl groups. Diets Plsm Brin zones Cortex Hippompus GSH + GSSG GSSG/GSH α-to GSH + GSSG GSSG/GSH GR α-to GSH + GSSG GSSG/GSH GR α-to C 13.0 ± ± ± ± ± ± ± ± ± ± ± 0.01 Cu 13.5 ± ± ± ± ± ± ± ± ± ± ± 0.02 Cho 12.7 ± ± ± ± ± ± ± ± ± ± ± 0.03 CuCho 15.8 ± ± ± ± ± ± ± ± ± ± ± 0.02 Results (men of 10 nimls ssyed in triplite ± SD) were otined s desried in Setions 2.9.1, 2.9.2,nd2.9.3.Totlglutthione(GSH+GSSG)ndGSHreexpressedsμmoles/mgprotein.GRtivityws expressed s U/mg prot min. α-to ws lulted in nmoles/mg protein. Results sttistilly signifintly different (P < 0.01) re indited with distint supersript letters (vlues within the sme olumn with different supersript letters re sttistilly signifint etween them).

8 8 Interntionl Journl of Alzheimer s Disese 4 Totl protein (ng/mg) d d d Cspse-3 (U/mg prot.) Cortex Control Cu Hippompus PGE2 Cortex Cho CuCho Hippompus PGF2α Figure 2: Prostglndin PGF2α nd PGE2 levels in rin ortex nd hippompus homogentes (ng/mg totl protein). Tretments re indited with different olors. Smples were nlyzed s indited in Setion Control (whiters), Cu (gryrs), Cho (drkgry rs), nd CuCho (lk rs). Results re expressed s men of 10 rts ssyed in triplite ± stndrd devition (SD). Signifint differenes were indited y letters (dt with distint letters re sttistilly different etween them t P < 0.01). 0 Cortex Control Cu Cho CuCho Hippompus Figure 3: Cspse-3 (U/mg totl protein) tivity in rin ortex nd hippompus homogentes. Smples were nlyzed ording to the proedure desried in Setion Tretments re indited with different olors. Control (white rs), Cu (gry rs), Cho (drk gry rs), nd CuCho (lk rs). Results re expressed s men of 10 rts ssyed in triplite ± stndrd devition (SD). There re no sttistilly signifint differenes (P < 0.01) etween tretments. enhned y Cu supplementtion nd even more so y simultneous tretment with Cu + Cho. The level of α-toopherol (α-to) deresed signifintly only fter CuCho tretment (pprox. 17% ompred to ontrol dt). In hippompus, the ehvior ws very similr, with the exeption of the inrement in totl glutthion ontent. Thus, hippompus homogentes showed signifint inrese in the GSSG/GSH rtio, tivtion of GR in Cu- nd CuCho-treted rts, nd. 34% derese in α-to ontent ompred to ontrol dt, with this ltter eing oserved only in the CuCho experimentl group Mrkers of Inflmmtion. In order to evlute whether simultneous supplementtion with Cu nd Cho lso produedninflmmtoryondition,wenlyzedthelevels of two proinflmmtory prostglndins, PGE2 nd PGF2α (Figure 2). In oth rin regions, osupplementtion signifintly inresed prostglndin levels. Interestingly, Cu nd Cho lone lso inresed prostglndins levels with respet to ontrol dt, nd ssoition of the two supplements produed n dditive effet Cspse-3 nd Clpins Ativities. We lso explored whether the prooxidtive nd proinflmmtory environment developed fter CuCho tretment ws le to trigger poptoti signls, to whih end we determined the tivities of the two min protese systems involved in progrmmed ell deth, spse-3, nd lpins (μ- nd m-). Cspse-3 tivity tends to inrese t lest in ortex homogentes, ut not to sttistilly signifint degree (Figure 3). Both lpin (millind miroisoforms) tivities inresed fter Cu tretment nd even more so fter CuCho supplementtion in oth rin zones (Figure 4) Biomrkers of Neurodegenertion. The onentrtion of the β-myloid peptides (1-42 nd 1-40) nd the Aβ 1-42/1-40 rtio in ortex nd hippompus re shown in Tle 4.The rtio (whih is the min inditor of neurodegenertive proess) ws different, depending on the rin zone exmined. In ortex, it ws found to inrese fter Cu nd Cho tretments nd to further inrese fter CuCho supplementtion. In hippompus, the Aβ 42/40 rtio inresed only in the CuCho experimentl group. There were no sttistilly signifint hnges of the rtio (1-42)/(1-40) in peripherl plsm. We lso investigted possile ltertions in the visuosptil lerning pilities through the Brnes mze test. We oserved minor (not sttistilly different) hnges in lteny to the first hole nd more sptil preferene for the espe region (holes 1, 0, nd 1) regrdless of tretment (dt not shown). Tken together, these hnges demonstrte minor ltertions in visuosptil memory suggesting tht simultneous supplementtion with Cu nd Cho produes n inrement in explortory tivity or sort of overexited ehvior ut with similr finl results to those oserved in the other experimentl groups. 4. Disussion Supplementtion of drinking wter with low mounts of inorgni Cu suh s those used in our experiments ws le to modify the sl sttus of redox iomrkers not only in peripherl plsm ut lso in the two zones of the entrl nervous system explored, ortex nd hippompus.

9 Interntionl Journl of Alzheimer s Disese 9 Tle 4: Aβ (1 40 nd 1 42) peptide onentrtions nd the rtio Aβ (1 42)/(1 40) in plsm nd in rin regions (ortex nd hippompus) in rts fed with the experimentl diets. Tretments Plsm Brin zones Cortex Hippompus Aβ (1 40) Aβ (1 42) Aβ (1 42)/(1 40) 10 2 Aβ (1 40) Aβ (1 42) Aβ (1 42)/(1 40) 10 2 Aβ (1 40) Aβ (1 42) Aβ (1 42)/(1 40) 10 2 C ± ± ± ± ± ± ± ± ± 0.12 Cu ± ± ± ± ± ± ± ± ± 0.24 Cho ± ± ± ± ± ± ± ± ± 0.15 CuCho ± 2.40 d ± 0.22 d 9.46 ± ± 2.03 d ± ± ± 1.44 d 8.82 ± ± 0.22 Results (men of 10 nimls ssyed in triplite ± SD) were otined s desried in Setion Individul peptides re expressed s piomoles/l (plsm) or piomoles/mg protein (rin homogentes). Results sttistilly signifintly different (P < 0.01) re indited with distint supersript letters (vlues within the sme olumn with different supersript letters re sttistilly signifint etween them).

10 10 Interntionl Journl of Alzheimer s Disese Ativity (μu/mg protein) Hippompus Cortex Hippompus Cortex m-clpins μ-lpins Control Cu Cho CuCho Figure 4: Clpins (μ- nd m-) (U/min mg totl protein) tivities in rin ortex nd hippompus homogentes. Smples were nlyzed ording to the proedures desried in Setion Tretments re indited with different olors. Control (white rs), Cu (gry rs), Cho (drk gry rs), nd CuCho (lk rs). Resultsreexpressedsmenof6rtsssyedintriplite± stndrd devition (SD). Signifint differenes were indited y letters (dt with distint letters re sttistilly different etween them t P < 0.01). Beuse of their short life spn, free rdils nnot e mesured diretly, for whih reson it is usul to nlyze the produts rising from retions used y retive speies with iomoleules. In line with this, we mesured the endoxidtion produts of lipids (TBARS) nd proteins (PCs) nd the prodution of NOx s iomrkers of prooxidtive tissue dmge. PCs inresed only fter CuCho tretment; however, TBARS nd NOx inresed fter sole exposure to tre mounts of inorgni Cu in drinking wter. The iomrkers ssyed were reproduile nd sensitive to the experimentl stimuli. Mny uthors hve ssoited neurodegenertive illnesseswithothlol(rin)ndsystemi(plsm) inrese in oxidtive stress [45 52]. Irrespetive of whether iomrkers re ustive ftors or whether they merely onstitute phenomenologilly ssoited hnges, they re useful tools for evluting the extent of dmge nd provide simple methodology for monitoring lrge popultions sujeted to environmentl Cu pollution or exposed to other risks ssoited with the development of AD. There is undnt evidene tht trnsition metls in generl, nd speilly Cu, re ustive ftors for oxidtive stress nd, s we mentioned previously, re strongly ssoited with the neurodegenertive proess [53 56]. The derese of α-toopherol in plsm nd rin zones homogentes nd the inresed GSSG/GSH rtio re oth mrkers of umultion of retive speies in lipid- nd wter-solule ell omprtments. The tivtion of GR n e interpreted s ompenstory mehnism of the enzymti ntioxidnt defense system in order to normlize the ltered GSSG/GSH rtio indued y otretment with Cu nd Cho. In greement with our results, Kojim et l. [57] hve demonstrted the indution of mrna oding for GR in the rin of mie irrdited with low dose of γ-rys. Also in line with this, we n speulte tht the oserved inrese in plsm totl glutthione levels my e due to ompenstory mehnism under the oxidtive insult evoked y the tretment studied here. This explntion is in greement with the suggestions of other uthors [58] nd my e the onsequene of n indution (overexpression) of ysteinyl-synthetse, the enzyme tht ontrols the iosynthesis of glutthione [59, 60]. The oxidtive stress indued y NCBC ould hve multiple effets on the signls sde tht depends on the redox stte nd n lso modify the tivity of ioni hnnels, trnsporters, nd enzymes. Pllottini et l. [61] oserved, in liver of Wistr rts treted with thioetmide, tht HMGCoAredutse overtivtion ws stritly relted to the mgnitude of the retive speies umulted. Our results demonstrte tht inorgni Cu supplementtion even t the low levels ssyed produed n inrese in ECho levels in ortex nd hippompus. Attriuting the inrese in Cho in rin to oxidtive stress-indued HMGCoA-redutse hypertivity ould e oversimplisti, nd this intriguing question remins to e resolved on the sis of new experimentl evidene. Cho in the nervous system (10-fold higher thn in ny other orgn) is minly unesterified [62, 63]. Furthermore, most of the Cho ontent in rin depends on the in situ iosynthesis tht ppers to e regulted y similr mehnisms oth outside nd inside the rin, with HMGCoA-redutse eing the most importnt regultory effetor [63]. However, the ext extent of Cho iosynthesis in neurons nd stroytes in vivo remins unknown [62], mking it diffiult to estimte the rel effet of the umultion of retive speies (NCBC-indued) on rin Cho iosynthesis. Interestingly, Choturnoverinindividulneuronsndstroytesmyin ft e very high nd reh n estimted 20% per dy, depending on the rin zone [62]. Unfortuntely, the diret effet of higher Cho levels in lood on Cho onentrtion in rin is diffiult to ssess. Cho trffiking etween rin nd peripherl lood implies the prtiiption of the loodrin rrier (BBB) tht hinders the diret pssge into or out of the entrl nervous system. However, for resons not yet understood, this restrition ppers to e redued in AD nd other neurodegenertive disorders [64]. Also, in ertin irumstnes, for exmple, in ses of vsulr injury due to oxidtive stress, the BBB ould e permele to intertion with the peripherl pool of Cho [64]. Moreover, reent experimentl evidene in mouse model of AD demonstrted tht inflmmtion is one of the key ftors in determining inresed BBB vulnerility [65]. Thus, from the ove, we n ssume tht peripherl Cho might ross the BBB into the rin, in prtiulr when the tissue is sujeted to oxidtive stress nd redox-indued inflmmtion s oserved in our experimentl model. Despite extensive reserh in reent yers, the role of ChosriskftorforADreminsontroversil[64], likely due to the still unresolved questions relting to the ext role of peripherl Cho in its level in rin. We n speulte tht NCBC indues nitrtive/oxidtive nd proinflmmtory onditions tht proly filitte endothelil

11 Interntionl Journl of Alzheimer s Disese 11 dmge nd indiretly modify BBB properties [64, 65], llowing peripherl Cho to enter the entrl nervous system. Oviously, gret del of experimentl work is still required to either onfirm or refute this working hypothesis. Severl unresolved issues rise douts onerning the enefiil effets of sttins in neurodegenertive ptients nd the notion tht high lood Cho is ssoited with rin dysfuntion. However, one possiility is tht the rel risk is the ssoition of hyperholesterolemi with prooxidtive nd proinflmmtory environment indued, for exmple, y NCBC. Brin Cho is involved in synptogenesis, the turnover, mintenne, stiliztion, nd restortion of synpses [63]. The funtionlity of synpses requires speifi lipid domins in neuronl nd xon memrnes whose omposition is ritil for the orret trgeting of the mjor memrne proteins, myelin iogenesis, ellulr differentition, signl trnsdution, nd mny other funtions tht depend on mirodomins nd speifi lipid rfts. The proportion of free ChotoEChointhesememrnedominsisruilftor in their iologil tivities [63]. For exmple, the enzymes responsile for the proessing of the myloid preursor protein (APP) to Aβ (β- nd γ-seretses) reside in Cho-rih lipid rfts of plsm memrne [66]. It ws suggested tht higher totl Cho/phospholipid rtio in ellulr memrnes ould ffet seretse tivities nd determine preferentil APP proessing pthwys [66], though in vitro studies suggest tht Cho might impir the trnsription of APP nd onsequently derese the vilility for its onversion to βa. However, it ppers tht this effet hs no signifint impt onthemountofprotein(thtstillexeedsthepityof the seretse system to proess it) nd only slight impt on the levels of the mrna enoding APP protein [67]. To dte, there re no findings eluidting the ext role of ECho nd how the rtio of free to esterified forms is le to modify seretse tivity nd other proess ssoited with the myloidogeni sde [63]. Zn et l. [45]suggesttht different soures of oxidtive stress, suh s NCBC, ould trigger the myloidogeni pthwy, whih my explin the higher Aβ 1-42/1-40 rtio we oserved in the rin ortex of Cu-treted rts nd in the group reeiving oth tretments (CuCho). The question s to how Cho nd Cu intert to lower the prodution of βa nd enhne oxidtive stress nd inflmmtion is diffiult to ddress. Experimentl evidene otined in ulture ells demonstrtes tht exposure of mrophges to CuSO 4, t level equivlent to NCBC in humns, indues SREBP-2 nd onsequently the expression of holesterogeni enzymes [68], thus tenttively providing new explntion for the pprently dditive effet we oserved etween Cu nd Cho. However, we were unle to find similr evidene in neuron or stroyte ultures, or even in experiments onduted on live nimls to explore these findings. There is yet nother possiility: tht the ssoition of the two supplements (Cu nd Cho) my ffet the lerne of βa nd filitte its umultion. Working with rits fed on diet with exess Cho nd inorgni Cu in their drinking wter, Sprks et l. [69] proposedthtchoused ltertions in the BBB ssoited with n inflmmtory ondition nd onomitnt overprodution of βa in the rin. In this model, Cu deresed the lerne of βa to the lood vi inhiition of LRP t the vsulr interfe [69]. Impired Chometolism,oxidtivestress,ndinflmmtionwerell ftorsssoitedwiththederesedlerneoftheβapeptide [63, 69]. In ddition, Lu et l. [14] lso demonstrted tht Cu exertes βa myloid-indued neurotoxiity through TNF-medited inflmmtory pthwy. Though the ext mehnism(s) underlying ll these effets is still unknown, it is nevertheless widely epted tht the AD pthologil sde is likely to e 2-stge event where deposition of βa nd neuronl pthology (tuopthy, neuronl injury, nd progrmmed ell deth, or susequent neurodegenertion with synpse nd ognitive loss) re sequentil rther thn simultneous proesses [70, 71]. Our model likely represents very erly step in these suessive events sine the sreening of the dmges oserved in visuosptil memory reveled only slight modifitions. The Brnes mze test nlysis demonstrted tht ll groups disply lmost norml loomotor nd explortory tivities nd sptil memory retention. The ehviorl modifitions indite tht the nimls of the ontrol nd Cho groups were fullyletoquiretheneessryknowledgeforthesptil tsk through trining, wheres the Cu nd CuCho groups wereonlyprtillyletodosoorpresentedslightlerning diffiulties. Interestingly, these lerning diffiulties were more evident during the evlution trils of Cho nimls, whih explored holes very distnt from the espe-hole region. Nevertheless, Cho nd CuCho nimls oth showed minor degree (not sttistilly signifint) of defiit in lerning nd sptilmemorypilities. Plsm levels of βa, prtiulrly the βa 1-40/βA 1-42 rtio, re well-reognized iomrkers of spordi AD [72, 73] nd even inditors of erly stges of the pthology [74]. However, prt from its role s iomrker, umultion of βa peptide in rin is omplex phenomenon with multiple nd onseutive (sometimes unexpeted) onsequenes [75]. In greement with this, Tmgno et l. [76] demonstrted tht oxidtive stress indued y Aβ25 35 resulted in n erly, signifint, nd time-dependent genertion of free HNE (hydroxyl-nonenl) nd H 2 O 2. Also, other uthors reported inresed levels of oxidtive stress iomrkers fter Aβ exposure oth in vivo nd in vitro [77 79]. Our results re therefore in greement with those of other groups reporting tht oxidtive stress nd Aβ re linked to one nother. Apprently, Aβ n indue oxidtive stress [80, 81], nd prooxidnts suh s NCBC n inrese Aβ prodution [82 84] in the mnner of n utostimulted proess. The inresed levels of the two min proinflmmtory prostglndins (E2 nd F2α) re onsistent with the inflmmtory ondition hrteristi of AD [85]. This proinflmmtory nd prooxidtive environment triggers the tivtion of lpins, wheres spse-3 tivity ws not signifintly stimulted under our experimentl onditions. In previous ppers, we lso found suh dissoition etween the effets of inorgni Cu overlod on the reltive tivities of the two protese systems, whih t lest in vitro experiments pper to depend on the extent nd intensity of exposure to Cu overlod [86]. However, in view of previous experimentl evidene, we nnot onlude tht neuronl deth is tully

12 12 Interntionl Journl of Alzheimer s Disese ourring.sofr,sitoetl.[87] hvereportedthtthe tivtion of μ-lpins in AD rin is not neessrily onsequene of the endstge of neuronl degenertion nd my reflet more widespred metoli ltertion tht preedes nd ontriutes to neuronl deth. In ft, they oserved inresed tivity of μ-lpins in the ereellum of AD without ny inrese in the rte of deth neurons [87]. In line with this, Nixon [88]estlishedthtdifferentftors ould led to lpin tivtion triggering neurodegenertion in the erly stges of AD development. Moreover, Trinhese et l. [89] lso reported tht lpins hve mny sustrtes tht ould e ffeted in AD ptients ut do not neessrily led to immedite ell deth. They lso stted tht μ-lpins re present predominntly in synpses, whih is in greement oth with the ft tht Cu onentrtion is prtiulrly high (miromolr) in the synpti left [90] nd with the wellestlished synpti pthology in AD [90, 91]. Nixon [88] suggested tht inresed tivity of lpins during norml ging my lso promote the development of neurologil disorders nd impired lium homeostsis, oth of whih ouldimptontheroleofthistioninthefuntionof ellulr memrne reeptors nd metllosignling in rin [91]. Finlly, in disussing the vlidity nd/or limittions of our experimentl system, it is neessry to onsider the level of the supplementtion with Cu using orl dministrtion. Our experimentl onditions were sed on previous work [13, 14] nd resemle the Cu levels ommonly found s onsequene of involuntry exposure through ir, food nd wter pollution [16, 92 94], ingestion of dietry minerl supplements, exposure of professionls engged in grohemil tivities [6, 11, 95], neurodegenertive ptients nd their first-degree reltives, or femle users of Cu-sed intruterine devies [17, 19]. Studies performed in rts demonstrted tht Cu metolism nd homeostsis re essentilly identil to those in humns [96].Intermsofdosge,thertsfrom the groups supplemented with Cu reeived 1.06 ± 0.11 mg Cu/Kg/dy (inluding Cu quire from food nd wter ingestion).fromthevilledtinhumns,wenssume tht generl popultion re reeiving 0.16 to 0.98 EAR or RDA whih is equivlent to 0.3 to 1.5 mg Cu/kg ody weight [97]. Very proly, humns re exposed to severl types of Cu-sed ompounds of different hemil strutures with differenes in their physil stilities, soluility, sorption pities, life s times into the orgnism, nd mny other prtiulrities relted to their exretion or ioumultion rtes. However, only inorgni Cu should e dngerous for its prole role s ustive ftor for neurodegenertion [3, 6, 8 10]. Thus, we think tht there re lot of questions to e nswered efore drwing relisti onlusion out the omprisons etween our experimentl onditions nd thetulhumnexposetocu. 5. Conlusions In onlusion, this in vivo study revels tht the ssoition of inorgni Cu nd Cho gives rise to prooxidtive nd proinflmmtory environment more pronouned thn tht produed y Cu nd Cho dministered lone. As desried efore, the omintion of these two ftors is ommon in mny humn popultions. We suggest tht the iohemil hnges oserved, in prtiulr, oxidtive stress, inflmmtion, nd the higher Aβ 1-42/1-40 rtio in the ortex of rts fed on Cu + Cho (CuCho), ould onstitute the initil stges of the development of neurodegenertive disese. In view of the undnt evidene of distured Cu homeostsis in AD [7, 56, 98, 99], we strongly reommend more indepth studies on the mehnism(s) responsile for the proneurodegenertive effet(s) of the ssoition etween NCBC nd Cho. Furthermore, it is reommended tht the present experimentl evidene e used to promote the investigtion of the emerging iomrkers suh s those exmined in this work to e pplied in peripherl plsm s preditive tool(s) in high-risk popultions. 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16 MEDIATORS of INFLAMMATION The Sientifi World Journl Gstroenterology Reserh nd Prtie Journl of Dietes Reserh Interntionl Journl of Journl of Endorinology Immunology Reserh Disese Mrkers Sumit your mnusripts t BioMed Reserh Interntionl PPAR Reserh Journl of Oesity Journl of Ophthlmology Evidene-Bsed Complementry nd Alterntive Mediine Stem Cells Interntionl Journl of Onology Prkinson s Disese Computtionl nd Mthemtil Methods in Mediine AIDS Behviourl Neurology Reserh nd Tretment Oxidtive Mediine nd Cellulr Longevity