View metadata,citationandsimilarpapersatcore.ac.uk Tech Francisco Ciruela, PhD Ciruela, Francisco This article isprotected Allrights bycopyright. reserved. doi: 10.1111/nan.12386 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen +34 93 4035808;email: [email protected] Phone: Spain; deLlobregat, sn, 08907 Feixa Hospitalet Llarga carrer University Hospital, Bellvitge Anatomy, of Pathologic Neuropathology, Service of Ferrer, Institute Isidro Prof. author: Corresponding CA: Short Locuscoeruleustitle: at early ofNFT stages pathology Spain deLlobregat, L’Hospitalet 6 5 4 deSaludCarlos Spain; Instituto III, Neurodegenerativas, 3 de Spain; Llobregat, L'Hospitalet 2 de Spain; Llobregat, L'Hospitalet 1 MScPol Andrés-Benito, pathologytangle at asymptomaticLocus coeruleus early a Article type : Original Article 19-Jan-2017 : Date Accepted :02-Jan-2017 Date Revised :08-Nov-2016 Date Received Universitat de Barcelona, L’Hospitalet de Llobregat, Spain; Spain; deLlobregat, L’Hospitalet deBarcelona, Universitat Institut de Neurociències, Universitat de Barcelona, Spain; de deBarcelona, Universitat Neurociències, Institut IDIBELL, Experimental, i Patologia Terapèutica Departament deFarmacologia, Unitat de Centro Investigación CIBERNED, enBiomédica Red de Enfermedades deBarcelona, Universitat Experimental, de i Patologia Terapèutica Departament deBellvitge, Universitari Hospital Patològica, Servei d'Anatomia deNeuropatologia, Institut

Accepted de Serveis Cientifics de (BT-E), Biologia Barcelona, I Universitat Tecnics, Unitat Article 1 , Luis Alberto Escobar, MDLuis Alberto 4,5 1 , Isidro Ferrer, MD, PhD Victor, Fernández-Dueñas, PhD 1 , Benjamín Torrejón-Escribano, PhD Torrejón-Escribano, Benjamín on and undergone full peer review buthasnot review undergone fullpeer on and nd middle Braak stages neurofibrillary Braakstages nd middle of Version of Record. PleaseVersion cite thisarticle as 1, 2, 3, 5, CA 5, 3, 1, 2,

4,5

, Margarita Carmona Margarita, 6 , Ester Aso,PhD Ester provided byDipositDigitaldelaUniversitatBarcelona brought toyouby 1,2 , CORE protein small RNAs nucleolar metabolism, protein Dopaminergic/noradrenergic denervation andDopaminergic/noradrenergic increased expression of accompanying NFT at early andformation asymptomatic stagesmiddle of NFT pathology. Conclusions amygdala. hydroxylase, Key words: and impairment clinicalevidence cognitive depression. before of compensatorysuggesting decreased adrenergic of occurring face input activation inthe receptor in hippocampusthe and amygdala occur stages at of NFTfirst pathology, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby These were accompanied by increased microglia and truncated were containing tau and VDACin neurons found. decreased tau, phosphorylated neuroketalincreased adducts and decreased superoxide 1in dismutase withneurons hyper- Results of NFTstages I-IV pathology were used. asymptomatic andamygdala individuals inhippocampus of inacohort at western blotting Methods pathology. (NFT) tangle of neurofibrillary stages amygdala to the earlyprojections asymptomatic andhippocampus at Braak andmiddle Aims Abstract andAWT and PCIN-2013-019-C03-03) (SBO-140028) FC. to by was it MINECO (SAF2014-55700-P alsosupported PA-B; to fellowship and IFI15/00035 –Fondos away and III FIS FEDER, to PIE14/00034 buildEurope: IF,Carlos PI14/00757 to deSalud studyThis was deEconomía Instituto Ministerio byy funded Competitividad, Funding data.No relevant disclosure and ofFinancial conflict interests Acceptedand increased immunoreactivityhydroxylase was inneurons LC, (TH) of the preserved decreased but TH Nucleolarof the Small atstage RNAs IV whenfamily, with compared stage I. Tyrosine and down-regulationmetabolism; of proteins DNA-binding coding andgenes for of members associated and proteinwith associated proteins shock genes binding ATP heat for with TNF- Article α : The present : study analyzes of characteristics molecular the locus coeruleus and(LC)

gene expression. arrays transcriptome gene Whole of revealed up-regulation coding genes : NFTs in : NFTs LC co-localized expression inparallelthe of with tau-kinases; increased : Immunohistochemistry, whole transcriptome arrays and RT-qPCR in LC, and arrays andRT-qPCR inLC, whole transcriptome Immunohistochemistry, : : Complex alteration of several metabolic pathwaysLC inthe of several metabolic occurs : Complex alteration Alzheimer’s disease, locus hippocampus, disease, Alzheimer’s coeruleus, amygdala, tau, tyrosine α α 2A 2A 2A 2A adrenergic receptor, mitochondria, transcriptome, heat shock proteins, heatproteins, shock transcriptome, mitochondria, receptor, adrenergic adrenergic adrenergic proteinreceptor levelswere found in the hippocampus

AIF1

,

CD68 , PTGS2 , α 2A 2A IL1 β adrenergic , IL6 and and the and the percentage ofpercentage neuron loss of about 70% atterminal stages of the disease [33, itis 36], decadesadvanced of 32-37]. ADhasbeenrecognized Considering stages a[14, for LCThe is the main noradrenergic nucleus affected inAD of [31]. Involvement the LC at autoreceptors. through themselves including leads adrenoceptors of theinhibition to inhibition, noradrenergic neurons conceivable many that functions byregulated the LC are severely dampened in advanced This article is protected by copyright. All rights reserved. copyright. This articleis protectedby is somethere evidence that released the LCactsNoradrenaline atfrom synapses specific through 22-28]. andneurogenesis [14, survival, ofand learning, bloodregulation flow, mo states, suchas wake-sleepfunctions memory cycles,cognition, arousal, emotional attention, insuperior display neuronal LCasahubnucleusimplicated networks Allthese the [21]. brainstem tegmentum lateral [20]. LC The receives input from various regions of the brain the from receivesalso projections thewhereas amygdala [19] cortex cerebral the innervating regions [15-18]. mainlyThe LCis hindbrain onlyinnervates nucleusthe noradrenergic structures suchas mostly theforebrain hippocampus cortex, andcerebral caudal portionthe in neurons nervousthe central system 14].[13, rostral portion of the LCinnervates the While The locuscoeruleus (LC) isa pontine nucleus containing the largest group of noradrenergic type [12]. Alzheimer 12]. [11, process) However, 25%of population the age of at 85the suffers dementia from of andsevere impairment cognitive dementia of (clinical stages the neurodegenerative with yet of only 5%have AD, to early about consistent changes reached leading thresholds the same way about85% inallindividuals; changes of Clinicalandpathological clinicalsymptoms appearance indo notprogress [8-10]. of most the brain alongwith disease progression paralleling the[4-7] progressive distribution which to haveof pattern extends aparticular alterations These morphological senileplaques [1-3]. of neuropil and (NFTs), tangles threads, component of neurofibrillary major neuritesdystrophic accumulationintraneuronal of hyper-phosphorylated and truncated 3Rtau and 4Rtau as the and senileplaques; of by morphologically accumulation a progressive neurodegenerative lasting disorder decades whichfor ischaracterized disease (AD), common is the inthemost elderly, Alzheimer's cause of impairment cognitive Introduction Acceptedadrenoceptors [29, 30]. Activation of Article β -amyloid deposits inblood-amyloid deposits vessels andto leading amyloid angiopathy; β -adrenoceptors are -adrenoceptors In activation of contrast, also excitatory. β -amyloid in the neuropil forming fibrillar diffuse deposits diffuse fibrillar neuropil forming in the -amyloid α 1 -adrenoceptors generally leads to and excitation, tor coordination, neuroinflammation, neuronal neuroinflammation, tor coordination, of thepopulation aged 65 has brain lesions

α 1 -, α 2 - or α β 2 - - This article is protected by copyright. All rights reserved. copyright. This articleis protectedby agewomen, 66.7 ± 6.68 years; stage n II: Accepted= 19, 12 7men, women, age 70.3 ± 7.83 years; of casescategorization was stage Braak as NFT I follows: of pathology: n =21, 10 11 men, Two of series cases were used. For andimmunohistochemical morphological studies, agonic state, hypoxia,evident long seizures were or excluded. study. Only cases with concomitant vascular mild small disease were accepted. Cases with senileplaques of Cases with[44]. pathologies were combined excluded the presentfrom adapted to paraffin sections 43],phases[4, Thal for andBraak NFTsfor Braak the staging was diagnosis following made Neuropathologic have beenmight present without beingrecorded insome individuals. andlear of attention, impairment disturbances, changes suchneuropsychological as recent in modifications wake-sleep cyclemood, and exhaustively to neurological notdirected were ‘minor’ analyzeclinical histories that duringobservation the post-mortem study. neuropathological itisworth However, stressing diseases. NFT hospital from variousthe non-neurological pathology was anincidental was reflected in not the of anyclinical records case. Allof werethem admitted and diedin admission depression hospital; of to the the welltime at preserved ascognitivelycategorized complaints; seriesNo individualsinthe hadneurological all of present werethem Hospital-IDIBELL. University Bellvitge and of localethics approval the the committee the 1716/2011) of AutorizaciónDecreto y deBiobancos, Funcionamiento Ministry Science of and Innovation: (Real on matter this Spanishlegislation of the guidelines the biobank) following ICO-IDIBELL Article brain Human samples were obtained of from the Institute BankNeuropathology Brain (HUB- Human brain samples andMaterials methods withassociated early stages of NFT pathology using whole arrays. transcription expression pathways altered identify gene and3: to unveil inLC altered amygdala; and LCon noradrenergic expression the receptors of the and inthe hippocampus TH andlocalsynapses assess of LC; of impact 2: thesealterations immunoreactivity, inthe the oxidative and responses, stress hydroxylase metabolism, tyrosine involved inenergy (TH) configurationphosphorylation, and truncation, associated expression of tau proteins kinases, stages ofBraak NFT pathologyinan to 1: attempt define characteristics of tau Based on this scenario, present studythe was onfocused the LC atthe asymptomatic first in the context ofNFTs AD-related [38-42]. pathology brain showing of the regions nuclei,isonethe withfirst raphe LC,together the However, AD. ning capacity. Therefore, these symptoms ning these capacity. Therefore, β -amyloid score and-amyloid CERADstages[5],

visualized with diaminobenzidine andH diaminobenzidine visualized with DK) 30peroxidase (Dako, for atmin room temperature. peroxidaseThe was reaction withincubation the primary antibody, the sections were incubated with EnVision +system with oneof Following inSupplementary listed overnight II. Table antibodies the primary followed 3%normal by horse solution.serum sections wereThen the at 4ºC incubated and without neuropathological lesions (no of casesCategorization was individuals with as noclinicalsymptoms middle-aged follows: hippocampus and Each amygdala. of these studies was carried inout allcases. peroxidases were blocked byperoxidases in10%methanol-1% were H incubation blocked buffer were antigenicity. boiledincitrate sections Endogenous(20 tau min) to retrieve 4 De-waxed sections, Immunohistochemistry pathology. 0 score plaque 0 phases varied 1 CERAD to to 1 andneuritic in caseswithfrom from NFT pathologyThal noNFT usedascontrols. pathology comparedwith MA withNFT individuals of purpose the second series was to assess changes inLC atearly and stages middle of delaymortem was 6.42 ± 3.64 hours (between 2 hand 14 h) (Supplementary I). Table The 4 men, n=5, IV, stage and ± 9.20; 76.7 age women, 2 6men, 8, n = III: stage ± years; 65.5 9.60 age 3women, 1 men, n=4, II: stage 610, 4women, agemen, 51.3 ± 5.9; stage nI: This article is protected by copyright. All rights reserved. copyright. This articleis protectedby andhydroxylase (TH) tyrosine protein levels of of anddetermination validation inLC, andRT-qPCR arrays second ofThe series cases whole was biochemical including transcriptome studies usedfor cases withoutcontrol NFT pathology were necessary. not purposeThe of the wasseries first to analyze linked changes to tau phosphorylation inLC; 77.75age ± 8.01 years. post-mortemThe delay was 7.4 ± 5.1 hours (between 2h and 21 h). n 5= 15, 77.6 III: men, stage age 10 women, ± andstage n=12, IV, 4women, 6.83; 8men, stained withstained DRAQ5 Acceptedfluorescence secondary antibodies against the corresponding hostspecies. Nuclei were were incubatedsections with Alexa488 Alexa546 Probes, or US) Molecular (1:400, 4ºC with overnight at washing, combinations primaryincubated the antibodies. After of and bodies, then rinsed ethanol and in70% water. washedThe sections indistilled were incell lipofuscin 15 present of autofluorescence DE) block min granules for the to (Merck, B solutionSudanblack were 4microns of thick, asaturated stainedDe-waxed with sections, Double-labeling immunofluorescence and confocal microscopy antibody. of the primary antibody; no signal was with obtainedincubation following only secondary the Article β -amyloid deposits inLC-amyloid deposits were not found in any cases. TM μ (1:2,000, Biostatus, GB). After washing, the sections were mounted m thick, of thick, LCwerem the processed immunohistochemistry. The for α 2A 2A adrenergic receptoradrenergic ( 2 O 2 . Control of the immunostaining included omission the immunostaining Control of . β -amyloid deposits, NFT NFT deposits, 0)calledMA:stage -amyloid n = = 78, 1women, agemen, 66.2 ± 6.45 years; 1 women, age 83.8 ± 5.93 years. post- The α 2A -AR) with western blotting in the blotting -AR) with western

2 O 2 solution (15 min) (15 solution Immunoblot. Plasmids Plasmids usedand transfection verify characteristics ofImmunoblot. to the (Thermo Fisher Scientific, Inc., Rockford, IL, and80 Rockford, USA) IL, Scientific, Inc., Fisher (Thermo concentration Protein previously was [47]. using determined the BCA protein assay kit This article is protected by copyright. All rights reserved. copyright. This articleis protectedby and7.4) 10mM MgCl 30 atfor centrifuged min 12,000xg. The weremembranes dispersed in50mM HCl(pH Tris washomogenate 10 centrifuged minfor at 1,000xg. was supernatant The resulting periods a of USA) three 10seach.for Polytron using Molecular (Roche Systems, The pH HCl, 1 7.4, Tris 300 mM containing cocktail aprotease EDTA, mM KClbuffer inhibitor Frozen samples of the hippocampus and amygdala were homogenized in ice-cold 10 mM Total membrane preparation of Alz50). neurons P-tauThr181-positive percentage co-expressing because protein the lessabundant protein represented a subset of the (i.e., former assessed protein. The werevalues expressed as the percentage of the abundant more varied on depending cells counted the bearing size population abundant of the the most in antibodies five selected per sectionfields and three sections inevery case. number The of expressing both antigens in relation to the of number cells stained with each one of the confocal the examined with microscope was assessedby counting the of number cells antibodies andwith specific two labeled proteins co-localization estimation of of The atp < 0.05;were statistically significant p considered < and 0.01; p < 0.001. and comparison test, differencesfollowedvariance multiple Tukey’s post-hoc (ANOVA) by MeanNFT pathology. values each for group were with compared one-way analysis of number LCneurons total atstages andIV to in of III, the of withII, I, neurons relation NFTs neurons were 5,360 Results countedAbout were intotal. percentage expressed asthe of implications insubstantial the purposes at hand, as discussed below. seriespresent as tissue didnotcontai blocks Unbiased stereological estimation of the number total of neurons [46] was not possible in the in sameestimated the which sections, haematoxylin. counterstained werewith slightly by 50 separated neurons x200; a the wasmicrons of number of at total magnification seriesall casesof the (n using morphological = 67), per non-consecutivethree case sections Neurons [45]. with ventricle the as IV NFTs revealed with antibody in AT8were the counted at tegmentum the level of the lower of segment aqueductthe cerebral of before just Sylvius pontine was in of part the rostral carried Quantification out LCwithin dorsolateral the the of studiesmorphological Quantification were examinedSections with aLeica confocal TCS-SL microscope. sealed,and overnight. dried US), Biomedicals, (ICN medium mounting in Immuno-Fluore Accepted expressed shown inHEK-293T cells are data inSupplementary 2. Article 2 , washed, and, re-suspended in the same medium as described n the whole LC. However, this limitation hadnon the this whole limitation However, LC.

μ g of protein were of used for g

α 2A -AR This article is protected by copyright. All rights reserved. copyright. This articleis protectedby absolute fold change >1.2 in with combination an p-valueunadjusted one-way they were showed expressed if considered(ANOVA Genes differentially test). an a or clustering co-expressed t-test) between method twofor classes(Student’s genes basedselected upon values experimental their using a test expression differential for in analysisconsidered batch aswellthe experimental (1 or asthe 2). Genes were first and were plaques (M neuritic or gender Cofactor W) USA). CA, Clara, (Santa software data the wasfor non-coding regions performed Affymetrix using Array Console Transcription of genes [49]. environment packagesraw for Processing Bioconductor in Rprogramming data were andMicroarray normalization, performed control, usingquality filtering Microarray analysis data Eberwine RT-IVT or the [48]. method preparation wassample based on the original T7 shown hybridization of microarray are Details in data 3. Supplementary ofThe method RNA using detected the softwareGraphPad QuickCalcs (p< 0.05). wasexpression probes. were studies different by using outliers tested RT-qPCR The values are presentednumber) in of such Supplementary Suitability casesI. Table mRNA for Spectrophotometer (Thermo Fisher Scientific aNanoDrop™ was using RNAconcentration assess RNAquality; evaluated CA,USA) Santa to Inc, Bioanalyzer withTechnologies Clara, the Agilent (Agilent determined and (RIN) RNAintegrity 28S/18S number were Hilden, GE). GmbH, ratios Qiagen® of LCwas supplierRNA from frozen (Rneasy the instructions the Mini extracted following Kit, andhybridizationSynthesis ofcRNA Whole-transcriptome arrays Spain). Amersham Europe 600 Barcelona, the Healthcare Imager GmbH, (GE bandsimmunoreactive were developed using a detection chemiluminescent andkit (Pierce) (1:30,000) or anti-rabbit IgG(1:30,000). anti-goat Theconjugated rabbit goat semidry systemtransfer and immunoblotted wi 10% polyacrylamideusing Proteins were gels. PVDF to transferred membranes a using was formed per- (SDS/PAGE) electrophoresis dodecyl gel Sodium polyacrylamide sulfate electrophoresis andGel immunoblotting (IPA) andMicroarray Ingenuity ontologypathway post-analysis: Gene (GO) data analysis valueschange between and 2.0 2.5 [50]. expressed obtainedgenes from the microarray were than lower currently selected fold analysis against Geneanalysis (GO) against through ontology packages bioconductor in R programming Accepted candidates wasputative analyzed using significance a biological for enrichmentgene a ofOnce differentially list expressed was genes selection identified from microarrays, the of Article th indicatedantibody,th andthe thenwith HRP- , Carlsbad, CA, USA). RIN (RNA integrity RIN , integrity CA,USA). (RNA Carlsbad, in vitro

transcription technology known as technology known transcription ≤ 0.05, as differentially asdifferentially 0.05, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby and *** p<0.001. Tendencies were at considered # p<0.1. betweenDifferences groups were at considered * significant statistically p<0.05, ** p<0.01 bypost-hoc test(ANOVA) when comparison followed multiple Tukey’s necessary. were unpaired student or with compared one-wayt-test two-tailed variance analysis of association betweenpossible linear two variables in continuous the Data studied samples. errorstandard of the mean Pearson’s (SEM). methodcorrelation was used to assess a wasfold-change determined usingthe equation 2 sample minus the mean post-mortemhuman brain 52]. [51, house-keeping was of genes Selection made on basisof preservation the their levels in used Fisher in study probes inSupplementary(Thermo the Scientific) III. listed Table are TaqMan Biosystems). (Applied System Sequence 7900 Detection ABIPrism HT the utilizing diluted reaction GE) 1:10transcription in plates 384-well Biotech,Steinfurt, optical (Kisker assays obtained retro- induplicate were on cDNAsamples the conducted RT-qPCR from real-timeQuantitative polymerase reaction chain (RT-qPCR) absence of out reverse DNA to transcriptase rule contamination. System thermocycler (AppliedOne Biosystems). RNAwas processed inparallel in the the provided andusing guidelines by manufacturer 9700USA) Gene the following Amp PCR Foster City, CA, out Biosystems, (Applied cDNAArchive withcarried the High-Capacity kit reaction ofRetro-transcription RNA samples selected on the basis of RIN valuestheir was polymeraseRetro-transcription reaction chain of microarrayValidation data used to data interpret the inthe context pathways, of processes, biological andnetworks. enriched. was USA) CA, Inc, System (Ingenuity analysis included inIPA core The function using p<0.05 as the cut-off point to determine whether GO database was significantly Accepted Data as were presented software. Inc.) mean PRISM Software, GraphPad ± (GraphPad were analyzedResults with statistically SPSS19.0 USA)(SPSS softwareInc., and analysis Statistical version 5.1. andData Visualization Software Analysis were group theanalyzed ANOVAwith one-way using Statistical test the Statgraphics Article ∆ CT of the population of control samples (calibrator samples). The samples). of of the population samples (calibrator CT control ∆∆ CT values were obtained from the - ∆∆ CT . Mean values ineach fold-change

∆ CT of each This article is protected by copyright. All rights reserved. copyright. This articleis protectedby d-f). of neurons 3A, stainedsubpopulation withantibody tau-C3 (Fig. Accepted antibody andanti-VDACtau-C3 discloseddecreased VDACimmunoreactivity inthe only decreased VDAC immunoreactivity 3Aa-c). immunofluorescence with (Fig. Double-labeling microscopy showed aminority hyper-phosphorylated LC neurons had that bearing of tau a marker of immunofluorescence mitochondrial Double-labeling andconfocal membranes. anion voltage-dependent against channelAntibodies immunostaining (VDAC) were used as Energy and metabolism, oxidative stress damage andresponses waskinases to restricted neurons with tau. hyper-phosphorylated in LC(Y279/Y216) GSK-P stage to I the several from to stage 2). Immunoreactivity IV (Fig. about AT8 and 65% of GSK-3 (Thr180/Tyr182), but80% of co-localization (Thr183/Tyr185), andand SAPK-JNK-P AT8 p38-P and of 100% and of AT8(100%) P-tauThr181 MAPK/ERK-P (Thr202/Tyr204), co-localization and confocal immunofluorescence co-localization showed microscopy Double-labeling total neuronsto those bearing hyper-phosphorylated tau (100% co-localization). 214) counted: 1C). in to (Fig. andubiquitin Immunoreactivity tau-C3 neurons was restricted in antibodies the LCatstages and numberanti-ubiquitin III IV of(total AT8-positive neurons that only 5% identified immunofluorescence of about AT8-positive neurons were stained with P-tauThr181-positiveneurons 128). counted: of number Double-labeling onlythat about5% neuron of P-tauThr181-positive revealed immunofluorescence Double-labeling 236). counted: neurons tauThr181-positive 80%about Alz50 neurons of with of P-tauThr181-positive number antibodies (total P- 100% of neuronsdisclosed that were antibodies stainedwith and P-tauThr181-positive AT8 Article acid421aspartic immunofluorescence (Fig. 1B). and Double-labeling confocal microscopy of neuronsminority were labeledwith antibodythe that tau-C3 recognizes truncated tau at at antibodiesAlz50 amino tau modifications with conformation-specific acids5-15, butonly a Most Ser396/Ser404 at phosphorylated of were PHF1). them (antibody with alsostained P-tauThr181, anddouble- anti-tau P-tauSer262, antibodies phospho-specific P-tauSer422, and deposition and3Rtau was tau-100 4Rtautau Abnormal with andwith positive antibodies about from progression NFT1% atstage Iof 15% pathology to about at 1A). stage IV (Fig of number and withThe total). AT8-immunoreactive increased age stage neurons relation to the number total of neurons per section 5,360 LC(about neurons counted in tau sites phosphorylation and Ser202/Thr205, expressed as the number of positive cellsin cerebral of aqueduct Sylvius inevery casestained with the antibody AT8which recognizes non-consecutive sections of the part of therostral LCat levelthe of the lower of segment the number ofThe neurons deposition hyper-phosphorylated tau waswith analyzed inthree first pathologyGeneral phosphorylation theLC: tau of and tau-kinases Results β -P (Ser9), and 42% (Ser9), andabout -P P-tauThr181 s were stained with antibody tau-C3 (total

This article is protected by copyright. All rights reserved. copyright. This articleis protectedby increased significantly expressiongene of showed arrays, the same RT-qPCR casesUsing transcriptome employed inwhole with morphology reactive short, thick varicose branches (Fig. 5A). a and Some cellsadopted of microglial NFT IV stages III at pathology. wasantibody, found Iba-1 number asrevealedwith inthe cells, microglial amoderate increase of contrast, numberThe of astrocytes was increased in not LCatstages not (data I-IV shown). In responses Glial withcompared LCneurons in without tau-C3 the same tissue section. on cellsurface dots when the immunoreactive of LCneurons tau-C3-immunoreactive counted:NFTs 28) showed adecrease of between 40% and 60% of synaptophysin- 126; counted neurons without with neurons NFTs: Quantitative 4B). total (total studies (Fig. cytoplasm neuronal were antibodies the stained with observed with inLCneurons tau-C3 Reduced synaptophysin-immunoreactive dots representing perineuronal synaptic contacts 4A). of NFT (Fig. pathology stages I-IV at in immunoreactivity phospho-tauNo modifications TH bearing inLCneurons were observed Tyrosine-hydoxylase andsynapses (TH) tau 3C, depositionphosphorylated (Fig. g-i). was immunoreactivity with receptor inLCneurons (EPOR) hyper- preserved erythropoietin AT8-immunoreactive neuronsdeposits assessed:(total 232) 3C, (Fig. a-f). Importantly, LC inneighboring immunoreactivity neurons lacking abnormal hyper-phosphorylated tau hyper-phosphorylated incontrast with containing tau all LCneurons preservation of andAT8 showedhemoglobin hemoglobin reduced anti- specific with immunofluorescence against antibodies double-labeling Finally, P-tauThr181-immunoractive neurons (total counted:antibodies 196). of inpracticallyimmunoreactivity the totality LC neurons stained with anti-P-tauThr181 at increased stages NKTstudies and immunoreactivity identified III IV decreased SOD1 and andconfocal immunofluorescence by 3B, microscopy d-f). double-labeling Quantitative (Fig. LC to as hyper-phosphorylated neurons(NKT) tau containing restricted revealedneuroketal same section was 3B,a-c). tissue byThis accompanied (Fig. increased expression of in when with tau deposition the neurons tau compared hyper-phosphorylated without with A decrease in superoxide dismutase 1 immunoreactivity(SOD1) was inLCfound neurons stages (Fig. 5B). 5B). (Fig. stages contrast, (which encodesCOX2), Accepted Article GFAP and IL10 IL1 mRNA expression levels were not inmodified LCat the same β , IL6, andIL6, TNF- α in andLC at III stages IV of NFTpathology. In AIF1 (which encodes Iba-1), (which encodesIba-1), α andhemoglobin

β / γ immunoreactivity in CD68 , PTGS2

Microarray analysis I-IV (Fig. 6B). whereas I, at expressionstages atstage was reduced TH increased significantly significantly expected, the anti- the expected, non-glycosylated to correspond of andglycosylated receptor, the As respectivelyforms [53]. α antibody was specific. the emptywith vector Figure lane:(Supplementary 1, mock), thus indicating that the several broad bands of ~46, 50,65 kDa Figure(Supplementary 1, lane: This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Data 4and FigureSupplementary Supplementary 2. and compromisesystem’, analysis ‘cellular is Detailed components’. shown in folding ‘protein mechanisms’, modification ‘post-translational identified functions cellular and disorder’, ‘hereditary ‘psychologdisease’, pathwayIngenuity analysisrevealed up-regulated pathways corresponding to ‘neurological RNAs (SNOR). nucleolar family Down-regulated were DNAbindingandgenes linked metabolism. to of members thesmall coded proteinsfor were linked to protein heatshockfolding, and protein ATPbinding, (p<0.05) wasdatabase and Up-regulated NFT pathology. genes I between IVof stages analysis enrichment analysis revealed the against most significant ontologygo Statistical analysisEnrichment database GO against and ingenuity pathway (IPA) analysis IV. Table in Supplementary is found of genes de-regulated list complete A (Fig. 7). heatthe map of at regulated representation significantly transcripts aprobability of <0.05 inLC betweenidentified stage andI stage IV of as inNFT pathology, graphically illustrated were profile in allsamples.gene were across Majorsequences differences detected to the analysisrelevant and therefore all samples were 6,870 included.A of total gene set nooutliers and were the detected, and application, filter normalization werecofactors not and only variables. gender associationThe was with age NFT After datastages. correlation showedmethod Pearson’s noassoci α the same samplesthe (Fig. 6A). expression The of and with lower progression, at III significantly declined with levels IVstages in age/disease butstage I, itwas decreased atstage significantly IV 6A). (Fig. Interestingly, TH expression the humanthe First, the specificity of the anti- specificity of the First, pathology 2A Accepted Article 2A -AR expression was significantly increased in the hippocampal hippocampal atmembrane inthe expressionfraction was significantly-AR increased -AR and TH expression levels hippocampus of levels NFTin andTHexpression andamygdala-AR I-IV stages at α 2A -AR -AR expressed inHEK-293Ttransiently cells. anti- The α 2A -AR didnotdetect any bands protein incells transfectedtransiently α 2A -AR antibody was validated with immunoblot detection detection ofwith antibody was immunoblot validated-AR ical disorders’. ical disorders’. analysisThe of and molecular ation among post-mortem post-mortem values, ation among delay, RIN α 2A

-AR inthe amygdala-AR was also α 2A -AR) which might which-AR) might α 2A -AR detected-AR In contrast, HSPA6 whenIV with compared stage II. Finally, stage I. of trend wasreduction significant alsoobserved for This article is protected by copyright. All rights reserved. copyright. This articleis protectedby LChas deposits inthe characteristicsphosphorylated tau the same as in those reported Accepted activatedExpression of involvedkinases intau with co-localizing phosphorylation hyper- number compromise thewhich donotsignificantly total of neurons in LC. the advantage ofstudy these hastaken observations onBraak NFT to pathology stagesfocus of numbers markedly decrease in the LC along with progression of dementia [62]. presentThe lossNeuron occurs in prodromalAD in cases with and impairment, neuron cognitive mild of AD stages [62]. celllossin preclinical LCwas at the showing small that insignificantly of method optical unbiasedstereological comprehensive the using study, fractionators, occurs atadvanced stages of AD 61].[60, This has beenrecently determined ina more unbiased studies methods using quantitative Seminal have shown neuron lossin LC that the categorized pathology ‘NFT use the descriptive term of asBraak stages’. For we herein toAD spectrum [59]. this reason, have term ‘AD’andpreferred the avoided (formerly with dementia only when tangles advanced) has alsobeen considered part of the Th pathology [58]. to tau were restricted lesions in plaques thosethe at brain (particularly NFT stages Iand and II, some at stage andIII), age-related primary rather tauopathy [56,57],because(PART) most had no However, 55]. it can be some[54, NIA/AA argued that cases not ADbut might represent ascategorized preclinical AD stages according to the ABCclassification proposed by the and to depression. related cognitive Allthese impairment symptoms cases major can be context ofthe early Braak staging of NFT pathology together with the absence of clinical present seriesCases inthe were on selected the presencebasis of ofthe in LCin the NFTs Discussion Article 8). (Fig. I stage IVstage when compared withI stage of significant inLCsamplesup-regulation confirmed RT-qPCR and II were study. includedinthe II, IV I, Stages DNA/RNA bindingproteins: 3. mechanisms protein 1. folding including: selected altered theWe main validation genes from functional pathwaysfive four for of microarrayValidation by data qRT-PCR SCARNA2 was significantly up-regulated atstage IV up-regulated when was with stage I. significantly compared ID4 was down-regulated at stage III when compared with stage I. A non- A I. stage with compared when III stage at down-regulated was was down-regulated inLCat was and stages down-regulated III, II, when IV with compared ID1 ; and; 4. Non-coding small nucleolar RNAs ;

DNAJB1 HSPA1B was also significantly up-regulated at stage up-regulated was alsosignificantly is is difficult to discuss at present, asPART discuss to atpresent, is difficult and ID4 DNAJB1 atADstage IV when with compared

; 2. ATP metabolism HSPA1B and DNAJB1 SCARNA2 β -amyloid ASPA6 at . ; model of frontotemporal lobar dementia linked to mutations in tomutations linked dementia lobar of frontotemporal model timingThe of these alterations is to similar that seen inP301S transgenic mice used as a deposition deposition progressive lossof synapticprogressive leading progressive inputs, to isolation. neuronal notion the reduced connectivitythat reinforcing with of neurons NFTs tomay bedue,inpart, havecontacts also been reported in neurons in tau-containing the hippocampus inAD [92], synaptic localization of inthis subpopulation of altered neurons.molecules Reduced synaptic accompaniedis seldom by synaptophysin suggesting immunoreactivityfocal clustering, synaptic only contacts alteration bearing Focal lossof is truncated inneurons tau. This found encodes COX2), expression by of mRNA accompanied increased are changes These morphological brain [89]. regions with pathology inother association tau andbuds, varicosities described as senescent a [88], alsoobservedmicroglia feature in inthe of with LCatmiddle pathology NFT short stages Microglia branches, appear ramified This article is protected by copyright. All rights reserved. copyright. This articleis protectedby cells catecholaminergic tyrosinedefenses and synthesis hydroxylase andfacilitates activity inperipheral andhomeostasis mitochondrial activity AD inneurons cortex deposition, aspreviously of tau phosphorylated the in cerebral reported and haemoglobin NFT pathology early inthe LCat oxidativeincreased stage damage suggesting reduced oxidative stress responses in the same neurons and in arguing favor of indicating oxidativeadducts, stress damage Hyper-phosphorylated tau deposition in LC neurons is accompanied by increased neuroketal thus accounting79], for numbersdecreased of inLCmitochondria neurons bearing tau-C3. produce [76- mitochondrial tau damage truncated tau andparticularly Hyper-phosphorylated only containing trunca inneurons was identified VDAC suggesting Decreased immunoreactivity, a decrease in the number of mitochondria, [75]. hasand tau truncation asbeen proposed aninducer of celldeath via caspaseactivation inneurons inAD[74], deposition acid421and aspartic truncation betweenat ubiquitin tau of ta includingrecruitment full-length formation, intau truncation neurons ataspartic acid 421 isneeded to develop thefully cascade of NFT taualtered conformation; tau truncation occurs only after complex tau phosphorylation, and cortex inAD Ascerebral phosphorylation described[63-66]. regions, inother of tau precedes Accepted biochemical responsesfrom differs of neurons and cells in mouse ofglial models Article [84] . hasIt been suggested that brain haemoglobin subunits may in participate redox [91] . IL1 β β / , γ IL6 isalsoreducedimmunoreactivity inLC neurons with hyper- [87] and . TNF- α . [85, 86] u association Strong [67-73]. has been found [80] ted tau as ted tau revealed with antibody tau-C3. the AIF1 , and reduced SOD1 immunoreactivity, . α (which encodes Iba-1), (which encodesIba-1), -haemoglobin modulates antioxidant antioxidant -haemoglobin modulates

MAPT [81-83] (FTLD-tau) (FTLD-tau) . Haemoglobin Haemoglobin . PTGS2 PTGS2 β [90] -amyloid (which and α

This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted [106-108]. of involvement The DNA bindingproteins and the snoRNA family pathology inthe splicing and alternative silencing assuch gene microRNA-dependent other among functions non-coding RNAs other of post-transcriptionalwhich processing RNAs, non-coding regulate RNAs including nucleolar (snoRNAs), transcription whosefactors activity dependson the binding protein partner which acts as helix-loop-helix protein of DNAbinding family of inhibitor member the inNFTs. protein and tau abnormal the folding [101-14] members protein shock of elements Representative these validated with clusters heatfour were RT-qPCR including when with stage I. compared andI; iv) members at down-regulated of the small nucleolarRNAs (SNOR) IVstage family proteins which atwere stage down-regulated DNA-binding when for IV compared with stage atstage whenwhichwereactivityIV up-regulated with compared stage iii) coding genes I; including metabolism, associatedgenes with ATPase activator activity andATPase regulator atstagewere up-regulated IV when with compared stage ii) I; associatedgenes with ATP with chaper linked proteinproteins folding, arrays deregulated transcriptome clustersWhole havei) including encodinggenes identified 1.2 combinedgreater than with p-value unadjusted consequence as a treshold those values that showed anabsolute fold change equal to or this reason we selected those showedtranscripts that higher the fold change, in establishing Article did not reach the high fold levels change commonly For transcript selection. established for obtained (DEG) genes differentially microarray expressed after The the raw processing data occur nuclei might atverytarget stages early Braak of pathology. NFT levels.protein observationsThe present show that compensatory changes in noradrenergic at amygdala ofstages NFTfirst pathology occurring inparallel with local decrease of TH present The showfindings expression levels increased of compensatory mechanism thefor lossof noradrenergic neurons [100]. isdecreasedhydroxylase plasma early a at stages suggesting in activity of AD, LCandhippocampusthe inadvanced of beta- stages 99]. dopamine Interestingly, AD[98, noradrenergicsprouting in afferents the hippocampus and [97], compensatory changes in Reducednoradrenergic AD[14, is innervation symptomatic by94-96]. accompanied innervationnoradrenergic andexpression of adrenergic receptors have been reported in at asymptomatic neurons Braak stages Reducedcortical I-IV. inLC immunoreactivity advanced stages of the disease 93].[14, The present showfindings preserved TH Decreased TH immunoreactivity andincreased neurons with NFTs in the LC occur inAD at . Altered expression Altered . of these isgenes unexpectednot as they likelyare to linked HSPA1A/B is, is, HSPA6 SCARNA2 one binding and heatshock bindingwhich protein (Hsp70, and 6), (Hsp70, member ID4 (small Cajal body-specific RNA 2), are RNA2), body-specific Cajal (small ≤ (Inhibitor of DNA binding 4) DNA binding of (Inhibitor 0.05.

α 2A -ARs in -ARs the hippocampus and DNAJB1 [105]

encodes a ( HSP40 . Small ) This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted 973 A JW H Budka, A.Ironside Boca Perry CRC London, New 2015: Raton, Press 858- York: Lowe R. In: Kalaria 3. Dementia. J, Press 2011: Neuropath 47-65 RO Basel: ISN Dickson, DW Weller. and pathology Themovement ofdementia disorders molecular Neurodegeneration: C, disease Neuropathology Duyckaerts Dickson DW. Alzheimer’s 2. andof In: its variants. 2006; disease. Alzheimer's history A. J natural andmolecular Delacourte of The 1. References pathology. NFT of in alterations the LCand in projections asymptomatic individuals early and at middle stages capacity, cannotlearning be excluded in this present observationsseries, unveil novel attention and impairment of andwake-sleep cycleand discrete in mood disturbances, compensatory activation inthe of decreasedface Althoughadrenergic input. subtle changes expressionincreased levels of Article LCbutof levels protein TH decreased in are the amygdala andhippocampus. Parallel immunoreactivityTyrosine RNAs hydroxylase inneurons (TH) ispreserved nucleolar family. of down-regulation codinggenes and proteins DNA-binding for members of the small and protein folding chaperone and binding, associatedgenes with ATP metabolism, and the LCat early stages of NFTpathology of includingup-regulation genes associated with andIL6, TNF- and increased expression with proliferation of concurs microglia haemoglobin of NFT pathology bystages accompanied reduced altered are intraneuronal mitochondria, and tau, misfolded incorporation of truncated tau in the LCat asymptomatic early and middle present hyper-phosphorylated andstudyThe of tau shows deposition intraneuronal that Conclusions nucleolus to the ribosome has been recently reported inAD[112]. inneurodegeneration participants [109-111], proteinand altered synthesis from the andnucleolar are RNAsandproteins asas nuclear LCinADisrelevant, of emerging key 9 : 187-194: α α . Whole transcription analysis. has transcription Whole shown metabolic pathways novel altered in and β , oxidativeincreased stress, and reduced stress responses. This α 2A Greenfield’s Neuropathology -ARs in thehippocampus and suggestamygdala

AIF1 , ninth edition. EdsSLove, edition. , ninth , CD68 , Alzheimers Dis PTGS2 , . Eds IL1 β ,

study of the BrainNet Europe Consortium. Europe Consortium. study of the BrainNet disease:a inAlzheimer's of neurofibrillary pathology H.Staging SB,Kretzschmar Wharton DR, N, Thal Streichenberger C, Stadelmann F, SeilheanD, Tagliavini Roggendorf W, E, Patsouris Monoranu Parchi P, Larionov D, C, Meyronet Kovács S, P, GG, Korkolopoulou A, King P,Kamphorst Ince G, Graeber, Giaccone W, I, Gelpi E, Ferrer K, Tredici 7. Alafuzoff I, Arzberger T, Al-Sarraj S Arzberger Al-Sarraj T, I, Alafuzoff 7. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted literature. the of Correlation Alzheimer disease a with changes neuropathologic review cognitive status: of JQ, DR, RL,Beach T, Troncoso JC, Sonnen JA, Trojanowski Thal TG. Woltjer Wisniewski MannMackenzieMasliah IR, DM, McKee Montine E, Morris AC, JC,Schneider TJ, JA, KA,Hyman Jicha BT, Iwatsubo T, Jellinger Kukull GA, LeverenzKövari E, LoveWA, JB, S, Davies Del K, Duyckaerts P, C,FroschTredici V, Haroutunian CM, HofMP, PR,Hulette Nelson C, EH,Bouras Bigio 10. Alafuzoff PT, I, Braak H,Cairns NJ, BJ, Castellani RJ,Crain JC. Morris W, R, Kukull BF,Higdon disease. C, Kurland Hulette R, of non-dementedNeuropathology aging: evidencepresumptive preclinical for Alzheimer J,Kurlan Kaye WR, LA, RC, Petersen Parisi JE, Dickson DW, Smith CD, Davis DG, M, C, RoeGrundman VD, CM, Buckles Xiong JL, McKeelDW, Price 9. FA, Markesbery Schmitt Hansen RC. 1095 Petersen H, Braak WC, LE, McDonald elderly. cognitively normal of Neuropathology KA,Petersen DW, Johnson Dickson GE, RJ, Smith M, Boeve Ivnik Floriach-Robert BF, Parisi Salviati A, Knopman DS, JE, 8. Article disease. sporadic Alzheimer's underlying H, Braak 6. process the pathological preclinical phaseof The Del K. Tredici AD. of development the relevance for its and DR,Rüb5. U, Thal Orantes M, Braak H. Phases of A in beta-deposition the human brain Neuropathol 4. Braak H, Braak E. Neuropathological staging of Alzheimer-related changes. Neurobiol Aging Neurobiol 1991;

J Neuropathol ExpNeurol 82 : 239-259 : 2009; 30 : 1026-1036 2012; , Brain Brain Pathol Bodi I, Bogdanovic N, Braak H, Bugiani O, Del O, Bugiani H, Braak N, Bogdanovic Bodi I, Neurology 71 2015; J Neuropathol Exp NeurolJ Neuropathol : 362-381 138 2008; 2002;

: 2814-2833 18 58 : 484-496 :1791-1800 :1791-1800 2003; 2003; 62 : 1087-: Acta This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted 1995 Berlin, L.20. Heimer humanThe brain and spinal cord, 2 humans. manipulations andpharmacological pathological alterations of locus coeruleus activity in and roles part of inthe regulation arousal its II: autonomic and physiological function Samuels ER, 19. locusSzabadi Functional of neuroanatomy the noradrenergic coeruleus: E. in neurons the primates. of prefrontal anthropoid cortex Sherwood Species-specific CC. distributions of hydroxylase-immunoreactive tyrosine MA, Raghanti 18. CD,Erwin MA, CJ, JM,Spocter Stimpson Hof Bonar Allman PR, JM, in amygdala the the rat. neurons brainstem to the nucleusand hypothalamic paraventricular the nucleus ofcentral and Petrov 17. T, Krukoff TL, serotonergic, Jhamandas JH.Branching of projections catecholaminergic noradrenergic, the of innervationdopaminergic of neocortex. Development Foote Morrison SL, JH. 16. and spinal their projections terminations. KN, 15. Westlund Article JD. noradrenergic Descending MG, Coulter RM, Ziegler Bowker ahead[Epub doi:10.1016/j.pneurobio.2016.04.001 of print] S0301-0082(15)30089-7. dis neuropathology inAlzheimer's Monoaminergic N, Bažadona D, Šimi 14. Buée L, de Silva R, Di Giovanni G, in coeruleus the ofregulation cognitive performance. CM, Wischik PR. Hof Usher M,13. Cohen D, Rajkowski JD, J,Aston-JonesServan-Schreiber G. roleThe of locus dementia. to inevitably leading Ferrer Defining12. I. Alzheimer as a common neurodegenerative age-related process not 960-969 disease:Alzheimer age categories from 1 to 100 years. Braak11. H, DR, Thal Ghebremedhin E, Del Tredic Curr NeuropharmacolCurr ć G, G, Babi ć M,Leko S, Wray CR, Harrington Delalle I, Jovanov-Miloševi Brain Res Brain 2008; Prog Neurobiol 1993; 6 : 254-285: Curr TopCurr Dev Biol Prog Brain Res 609 : 81-92: 2012; ease. Prog Neurobiol ease. Prog Neurobiol Apr 12. pii: 2016; i K. Stagesi K. of processthe pathologic in 97 Science nd

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This article is protected by copyright. All rights reserved. copyright. This articleis protectedby deposition intau neurones and cells, butnot with increasedglial nuclear DNA vulnerability with expression isassociated Phosphorylated early ERK2) map (ERK1, Ribalta T. kinase F, BlancoR, Rey Carmona Ferrer I, E, Puig B, M, Viñals 65. MJ, R, Goutan Cardozo A, Ribera 108 expressed intau depositsinneurons cells and intauopathies. glial andcalcium/calmodulin-dependent (CaM kinase II are differentially kinase II) (SAPK/JNK-P), protein ofkinase 38(MAPK/ERK-P), protein stress-activated kinase (p38-P), kDa Blanco Carmona Ferrer protein I, B. R, M,64. kinase Phosphorylated mitogen-activated Puig 60 and is related to tau phosphorylation butnot to apoptosis. Activation of the JNK/p38 pathway occurs indiseases characterized by tau pathologyprotein Atzori C, 63. B, Ghetti Srinivasan Piva R, disease:cortical subcortical? or Alzheimer's Arendt 62. T, Brückner MK, M,Morawski Jäger C, lossin Early neurone HJ. Gertz nucleusin the coeruleus. 61. Busch C,Bohl J, Ohm Spatial, tempor TG. nucleus coeruleus during aging. 60. Ohm TG, Busch C, Bohl J.Unbiasedestimation of neuronal numbers in the human M, Halliday K, Goedert Neuropathol Acta Tredici Del L, G, M,Neumann MG, Spillantini M,Tolnay disease. of PART ispart Alzheimer Uchihara T. JP, Buée C,Braak H,Brion Duyckaerts 59. Alzheimers Dis cases with disease. early Braak are stages Alzheimer’s of neurofibrillary pathology 58. Giaccone existence notallthe tauopathyG. of primarythat suggests The age-related differents from classical JP, a Vonsattel PART, JQ,tauopathy, JC, distinct T. Wisniewski Troncoso Trojanowski Montine NelsonPT, TJ, F, Schmitt Schneider JA, Serrano-Pozo A, DR, Thal JB, Toledo Hyman BT,Jack CR Jr, Jicha GA, Knopman DS, Kovacs Mackenzie Masliah GG, IR, E, KA, I, Alafuzoff J, Attems 57. Jellinger Beach NJ, Cairns DW, Crary JF, Dickson TG, Hof PR,

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ingrowth. ingrowth. in Alz human brain. II. axonal morphology BoozeMactutus97. RM, Davis Frequency CF, CR, Gutman JN. analysis of catecholamine disease. Alzheimer’s in D,Levey norepinephrine andWeinshenker mild AI. impairment cognitive Targeting B, Chalermpalanupap Kinkead Hu 96. T, KummerMP, Heneka Hammerschmidt MT, T, WT, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted 2006; postmortem subjects with Alzheimer’s diseaseand dementia with Lewy bodies. in changes noradrenergicthe nervous system inthe locusceruleus and hippocampus of MA. Raskind JL, Leverenz SS, Compensatory Szot P, Greenup ER, JB,Peskind 99. White Kamphorst IE, disease. in Alzheimer's J,Sommer MH, Gilhuis Botterblom MG, W, Eikelenboom Feenstra P, WJ, Swaab DF. Increased activity surviving of Hoogendijk locus ceruleus 98. neurons cortex. alphadecreased 2-receptors demonstrated by [3 Adrenergic RN,Andorn Kalaria AC. rece 95. Article and Down’s syndrome. and enzymes cholinergic Catecholamines inpre-senile dementia andsenileAlzheimer-type A. Yates CM, 94. Simpson onA, J, MaloneyGord IM, Urquhart Ritchie AllisonY, AF, disease. Alzheimer's coeruleus in degeneration neurofibrillary and early cognitive impairment aging, mild 93. Grudzien A, Shaw P, S, Weintraub E, Bigio Mash DC, inpatientsneurons disease. with Alzheimer's Mesulam MM. Locus A, Avila J, of phospho- DeFelipe J. The influence P, Merino-Serrais 92. Benavides-Piccione R, L, Blazquez-Llorca Kastanauskaite A, Rábano species. and plaques, tangles, oligomeric with signals disease inAlzheimer correlations mice: and APP/PS1 Neuroinflammatory transgenic 26 Neurobiol Aging : 467-478: J Neurol Sci Neurobiol Aging Alzheimer’s ResTher 1993; 1991; 1991; Brain Res Brain Ann Neurol 119 12 : 110-118: : 131-136: 1983; 1999; 2007; 280 45 2013; heimer’s diseaseheimer’s andhippocampal sympathetic :119-126 :119-126 28 : 82-91: Brain

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This article is protected by copyright. All rights reserved. copyright. This articleis protectedby neurodegeneration. Accepted Alonso JR, Valero Berciano J, MT, FC, Baltanás 110. pre- M. of signs Lafarga Nuclear linked DNArepair. to defective silencing gene scale and chromatin reorganization reveals large inpcdmice celldegeneration Purkinje AlonsoJR, Berciano E, MT, V, Lafarga Casafont I, FC, Baltanás Weruaga 109. M.Lafarga Anupdated human AR. snoRNAome. 108. Jorjani H,Kehr S, DJ, Jedlinski HertelGumienny R, J,Stadler PF, M,Zavolan Gruber regulation. innon-coding variations Genomic V. Bhartiya Scaria 107. D, and Structure, RNAs: function of small role RNAsinhumannucleolar Regulatory diseases. Stepanov 106. Komissarov Filippova Semenov AB, GA, EV,Richter JA, Kuligina VA, DV. 103 differentiation 4 cancer. development From (ID4): to Havrda MortonMC, Carey J, D, Inhibitor DJ, Chaudhary Patel J. 105. of Biomolecules chaperones and co-chaperones their in targeting misfolded proteins to the proteasome.Article A, Ravid Shiber Chaperoning 104. T. proteins diverse of Hsp70 destruction: roles for polypeptides. misfolded unfold catalytically 103. Priya S,Sharma P. SK, Goloubinoff chaperonesMolecular as enzymes that Top MedChemCurr Prins HH.DNAJ Kakkar LC, V, Kampinga 102. proteins and diseases. aggregation protein 291-300 Young Mechanisms101. JC. of Hsp70 the chaperone system. 99 disease. activity inAlzheimer’s D. Muck-Seler decreaseGenotype-independent inplasma dopamine beta-hydroxylase Mustapic M,100. Presecki P,Pivac N,Mimica Hof PR,Simic V, N, Folnegovic-Simic G,

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This article is protected by copyright. All rights reserved. copyright. This articleis protectedby manuscript. andsuggestions.comments Allof the authors to contributed the version of thefinal advanced versionfirst of the whichmanuscript among wascirculated allthecontributors for noradrenergic system; the study, IF of designed the andwrote supervised results, the the studies statitiscal anddata assessment molecular to FCdirected EA contributed processing; BT-E, on studies sections; immunohistochemical confocal quantitative microscopy studies. hippocamopus andthe MC,amygdala; immunohist studies in the molecular VFD, arrays andRT-qPCR out thestudy validation; carried of PA-B oftheContribution authors processingbioinformatics of and data, we Yohannan thank T. editorial help. for to thank Ferran Unitof Statistics and Briansó, VHIR, the preliminary for Bioinformatics, wish Spain. Barcelona, (VHIR), We Research Unit Institute (UAT), Vall d’Hebron Technology researchThe was approval. givenethical Microarrays were outat carried Highthe Acknowledgements 26 of protein synthesis infrom Alzheimer's: the nucleolus to the ribosome. LucasJJ, P,GilL, Garcia-Esparcia Hernández-Ortega K, 112. machinery Ferrer I. Altered characterization.transcriptional and molecular structural, chromatin domain: DNAinanovel unrepaired specific of Berciano MT, Mata-Garrido J,Casafont I, O, Tapia 111. Lafarga M. Neuronal accumulation

Accepted Article 593-605. :

Acta NeuropatholActa Commun ochemistry and tissue LAE, preparation;

2016; 4 : 41 Brain Pathol Brain 2016; This article is protected by copyright. All rights reserved. copyright. This articleis protectedby with stagecompared III. 0.01; &&&p and< ~p II; with <0.001 compared stage < 0.05; p ~~ p< 0.01; ~~~ <0.001 at$ p significant < 0.05; $$ p by post-hoc comparison andTukey’s test, multiple differences were considered statistically values each of comparedgroup were with one-way of variance analysis (ANOVA) followed revealed with antibody AT8 inLCat Braak stages II, andI, II of NFTIV pathology. Mean 1 Figure legends Figure (green) and(green) (Y279/Y216)GSK-P (red); c, f, (green)AT8 andp38-P AT8 (red); (green)j-l: and GSK-3 g-i: (red); and SAPK-JNK-P d-f:(green) AT8 (red); and MAPK-ERK-P (green) tauThr181 immunofluorescencerevealed by and double-labeling confocal microscopy. a-c: P- Figure 2

Figure 3 Figure bar = 50µm. (blue); P-tauThr 181 (red) in with Nucleistained 181 i: DRAQ5 LC.c, (red) P-tauThr merge. f, and (green) ubiquitin g-i: and (red); (green) P-tauThr181 tau-C3 (red); d-f: and P-tauThr181 usingmicroscopy of combinations anti-tau a-c: antibodies andanti-ubiquitin; Alz50 (green) bar = 25 h:Alz50; i: µm. tau-C3; PHF; a: antibodies: anti-tau tau 100;b: 3Rtau; c: 4Rtau; Accepted Nucleistained i:merge. c, withf, DRAQ5 whereas EPO bar(green), neurons; receptor (red) intaug-i: ispresent containing = 50µm. d-f: Hbneurons; alsodecreased beta (red) immunoreactivity inAT8-positive is neurons receptor;and erythropoietin a-c: Hbalpha (red) is reduced inAT8-immunoractive (green) (green) tauThr181 andneuroketal bar(red) inneurons; co-localize = 50 µm. immunoreactivity is P- in SOD1 d-f: withreduced a-c (arrow); neurons (red) P-tauThr181 bar c, = (red) tau-C3 arrow; 50µm. merge; f: containing P-tauThr181 d-f:neurons containing (red); VDACimmunoreactivity isdecreased inneurons Decreased a-c: of immunoreactivityis seeninaminority Mitochondria; VDAC(green) Article : Expression and localization of tau Expression andhyper-phosphorylated and: inLCaskinases localization of tau : : Mitochondria, oxidative: Mitochondria, stress and responses, and hemoglobin in LC. A ) Percentage of neurons Percentage ) containing hyper-phosphorylated tau deposition as B ) of neuronsImmunostaining intau-positive LC different using < 0.01; $$$p < 0.001 with compared stage &pI; < && 0.05; p C ) Double-labeling immunofluorescence Double-labeling andconfocal) TM (blue) (blue) i, l,o: Nucleistained with DRAQ5 merge. d: e:P-tauSer262; P-tauThr181; AT8; f: g:

B β ) and ) Oxidativeresponses; stress -P (Ser9) (red); m-o: P-tauThr181 TM (blue); bar = 30 µm. bar = 30µm. (blue); C ) Hemoglobin A TM )

probes areprobes with normalized combined mean values of pawith noNFT individuals andin middle-aged IV, Figure 5 This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Figure 4: along with cells diseaseprogression; withmicroglial short, thick, and varicose branches also Moderate increase in NFT pathology. numberof the of Iba-1- immunoreactive cellsis found disease progression. disease progression. responses is manifested by increased stage wereIII. consideredTendencies at#p Moderate< 0.1. inflammatory activation of < 0.001 compared with stage andII; ~p < 0.05; ~~ p < 0.01; ~~~ p <0.001 compared with $ p < 0.05; $$ p < 0.01; $$$ p < 0001 compared stage I;&with p < 0.05; && p < 0.01; &&& p considered at*statistically significant p < 0.05; ** p < 0.01; *** p < 0.001 compared with MA; by(ANOVA) followed post-hoc Tukey’s comparisonmultiple test and differences were Mean values each fold-change for weregroup compared with variance one-way of analysis XPNPEP1 encodes COX2), present. bar = 20 µm. Nucleistained with = DRAQ5 20bar µm. are (red); labeledwith NFT pathology; c:merge; dots IVdeposits remaining arrows; of stage synaptophysin-immunoreactive in dots containing(green) tau-C3-immunoreactiveneurons neuronsnegative atBraak stage IV of pathology; c: NFT bar merge; = 50 µm. isexpressed equally with(green) inneurons and immunoreactive phospho-tau (red) Accepted stages. Article : B) A . ADcases. analyzed are the same as those used inwhole-transcriptomearrays. Tyrosine hydroxylase Tyrosine andsynaptophysin inLCneurons. ) Immunohistochemistry to in (B), and to (c) Iba1 III I(a), thestages LCatBraak IV Immunohistochemistry ) mRNA expression of mRNA GFAP GFAP , IL1 β and , IL6,IL10, IL10 AIF1 expressionmRNA levels are not atmodified these and TM AIF1 (which encodes Iba-1), (blue). TNF- , CD68 α thology. Expression distinct values the thology. for in the locus at Braak stages I, II, II, and , PTGS2

AARS , IL β , A , CD68 GUS- ) TH immunoreactivity IL6, and , β , PTGS2 HPRT1, B TNF- ) Reduced (which α and with with II, and II, III, see IV; areTable 1) shown. of Quantification samples MA each individuals human representative middle-aged group I, for (i.e. andstages immunoreactive with bandswereThe Two different visualized chemiluminescence. (1 normalized with with normalized a rabbit polyclonal anti-TH antibody (1 immunoreactive bands corresponding to to bands corresponding immunoreactive andhippocampus (A) (B) amygdala membranes are shown lower The inthe panels. with SDS-PAGE and immunoblotted using andimmunoblotted a SDS-PAGE anti- with rabbit extracts (80 Membrane stages of NFT pathology. and Braak different hippocampus amygdala at the This article is protected by copyright. All rights reserved. copyright. This articleis protectedby 6 Fig. Figure 8: Figure and IV of NFTpathology. inLC showing atBraak III, expression profile stages gene II, differential I, transcripts array p at

Accepted Articleμ g/ml). A IgG(1/30,000) was HRP-conjugatedg/ml). anti-rabbit used as a secondary antibody. : Expression of alpha-2A adrenergic Expression adrenergic ofreceptor: ( alpha-2A , GUS- mRNA expression mRNA levels of μ Significant differencesgene areSignificant seen when gene comparing expression profiles β g) g) from human hippocampus (A) and amygdala (B) necropsies were analyzed , HPRT1, α -actinin expression. Data were expression. -actinin by expressionnormalized of the and XPNPEP1 SCARNA2 μ g/ml), andg/ml), a rabbit anti- . Cases. analyzed the same are as those used in α 2A AR ineach andTH human sample were B) , Hierarchical clustering heat of clustering map mRNA Hierarchical HSPA6 α 2A AR) and tyrosine hydroxilase (TH) in and hydroxilaseAR) (TH) tyrosine α

2A , AR polyclonal antibody (1 antibody (1 AR polyclonal ID4 α , 2A α HSPA1A/B, AR and expression inTH -actinin polyclonal antibody antibody polyclonal -actinin and DNAJB1 α 2A AR and μ g/ml), g/ml), in extracts were analyzedextracts with and SDS-PAGE immunoblotted This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Supplementary figure 1: pcDNA3.1+ vectorpcDNA3.1+ (mock). concentrations Increasing 2,(1, and3 diagram pathways. of altered Interaction diseases andbiofunctions Braak stage comparing with IV stage of NFTI pathology. SupplementaryA) figure 2: secondary antibody. immunoreactiveThe bands were chemiluminescence. visualized with antibody (1 polyclonal Accepted Article HEK-293T cellswerecells. with transfected transiently cDNAencoding the μ g/ml). A HRP-conjugated anti-rabbit (1/30,000) A wasIgG anti-rabbit HRP-conjugated g/ml). usedas a Immunoblot detection of human detection of Immunoblot IPA analysis of top altered networks in LC and network-related inLCand networks network-related top analysis altered of IPA

α 2A -AR expressed in HEK-293T inHEK-293T expressed -AR

using a anti- rabbit μ g) of cellmembrane α 2A -AR or empty-AR α 2A

-AR B)

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This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article

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